Leukemia (2006) 20, 911–928 & 2006 Nature Publishing Group All rights reserved 0887-6924/06 $30.00 www.nature.com/leu LEADING ARTICLE

Phosphoinositide 3-/Akt signaling pathway and its therapeutical implications for human acute myeloid leukemia

AM Martelli1,2, M Nya˚kern1, G Tabellini3, R Bortul4, PL Tazzari5, C Evangelisti1 and L Cocco1

1Cell Signalling Laboratory, Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell’Apparato Locomotore, Sezione di Anatomia Umana, Universita` di Bologna, Bologna, Italy; 2ITOI-CNR, c/o IOR, Bologna, Italy; 3Dipartimento di Scienze Biomediche e Biotecnologie, Sezione di Citologia e Istologia, Universita` di Brescia, Brescia, Italy; 4Dipartimento di Morfologia Umana Normale, Universita` di Trieste, Trieste, Italy and 5Servizio di Immunoematologia e Trasfusionale, Policlinico S.Orsola-Malpighi, Bologna, Italy

The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is the 1970s,1–3 and patients with AML arising out of myelodys- crucial to many aspects of growth, survival and apoptosis, plastic syndrome (MDS) or who are older than 60 years do even and its constitutive activation has been implicated in the both 4 the pathogenesis and the progression of a wide variety of worse. Therefore, there remains a need for new, rationally neoplasias. Hence, this pathway is an attractive target for the designed, minimally toxic, effective therapies for AML. development of novel anticancer strategies. Recent studies Several independent laboratories have demonstrated that showed that PI3K/Akt signaling is frequently activated in acute AML arises from leukemic stem cells (LSCs) that have an myeloid leukemia (AML) patient blasts and strongly contributes extended lineage negative (linÀ), CD34 þ , CD38À, to proliferation, survival and drug resistance of these cells. CD123 þ , CD33 þ , CD13 þ /À.5,6 Indeed, injection of these cells Upregulation of the PI3K/Akt network in AML may be due to several reasons, including FLT3, Ras or c-Kit mutations. Small into immunocompromised nonobese diabetic/severe-combined molecules designed to selectively target key components of immunodeficient (NOD/SCID) mice leads to a disease with this cascade induce apoptosis and/or phenotypic and molecular characteristics of human AML. Over markedly increase conventional drug sensitivity of AML blasts the last few years, remarkable progress has been made in the in vitro. Thus, inhibitory molecules are currently being devel- elucidation of the molecular pathogenesis of AML. A recent ‘two oped for clinical use either as single agents or in combination hits’ model has suggested that AML development requires with conventional therapies. However, the PI3K/Akt pathway is important for many physiological cellular functions and, in multiple genetic changes that deregulate different cell pro- 7 particular, for insulin signaling, so that its blockade in vivo grams. fusion such as AML1/ETO, might cause severe systemic side effects. In this review, we PML-RARa, CBFb/MYH11 or MLL/AF9 block myeloid cell summarize the existing knowledge about PI3K/Akt signaling in differentiation by repressing target , thus providing one AML cells and we examine the rationale for targeting this necessary event for leukemogenesis.8,9 Disordered cell growth fundamental signal transduction network by means of selective pharmacological inhibitors. and upregulation of cell survival genes is a proposed necessary Leukemia (2006) 20, 911–928. doi:10.1038/sj.leu.2404245; second event. Mutations in growth regulatory genes such as 10–12 published online 27 April 2006 FLT3, Ras and c-Kit are common in AML patients. The most Keywords: signal transduction networks; 3-phosphorylated inositol recent data provide evidence of great interdependence between lipids; apoptosis; drug resistance; targeted molecular therapy these two classes of molecular events. Indeed, changes in the transcriptional control in hematopoietic cells modify the arrays of signal transduction effectors available for growth factor Introduction receptors, whereas activating mutations in signal transduction molecules induce alterations in the activity and expression of Acute myeloid leukemia (AML) is a heterogeneous group of several transcription factors that are essential for normal myeloid malignant hematopoietic disorders characterized by uncon- differentiation.13,14 trolled proliferation of clonal neoplastic cells and accumulation The phosphoinositide 3-kinase (PI3K)/Akt signaling network is in the bone marrow of blasts with an impaired differentiation crucial to widely divergent physiological processes that include program which are blocked at various maturation steps and cell cycle progression, differentiation, transcription, translation resistant to cell death. Acute myeloid leukemia accounts for and apoptosis.15,16 It is targeted by genomic aberrations approximately 80% of all adult leukemias and its overall including amplification, mutation and rearrangement more incidence has been stable or slowly increasing over the last frequently than any other pathway in human cancer, with the 15–20 years.1 Despite considerable advances in the diagnosis of possible exception of the p53 and retinoblastoma pathways. the different AML subtypes and progress in therapeutic Activation of PI3K/Akt signaling results in disturbance of control approaches, current chemotherapies produce only initial remis- of cell proliferation and apoptosis, ensuing in competitive sion, so that most patients will relapse and die from the disease. growth advantage for tumor cells.17–19 Furthermore, it is now The 5-year survival rate of AML has hovered at 15–30% since clear that PI3K/Akt axis upregulation may be one of the major factors undermining successful antineoplastic treatment, thus Correspondence: Dr AM Martelli, Dipartimento di Scienze Anato- portending a poor prognosis in many cancer types.20 Therefore, miche Umane e Fisiopatologia dell’Apparato Locomotore, Sezione di the PI3K/Akt pathway is an attractive target for the development Anatomia Umana, Cell Signalling Laboratory, Universita` di Bologna, of novel therapeutic strategies in patients with various tumor Bologna 40126, Italy. E-mail: [email protected] types. Received 23 February 2006; accepted 27 March 2006; published Several recent papers have highlighted that the PI3K/Akt axis online 27 April 2006 is activated in AML.21–24 Remarkably, both the disease-free PI3K/Akt signaling in AML AM Martelli et al 912 survival and overall survival were significantly shorter in AML organs and a selective impairment of development, cases with upregulated PI3K/Akt pathway.25 Therefore, this respectively.34,35 signal transduction cascade may represent a valid target for Akt contains an NH2-terminal pleckstrin homology (PH) innovative therapeutic treatments of AML patients. The main domain, which interacts with the phosphorylated lipid products aim of this review is to discuss potential antineoplastic strategies of PI3K (mainly PtdIns (3,4,5)P3 and, to a lesser extent, targeting this signaling network in AML. However, we shall phosphatidylinositol 3,4 bisphosphate (PtdIns (3,4,)P2)) synthe- begin with a general outline of the mechanisms that govern sized at the plasma membrane. Akt recruitment at the plasma PI3K/Akt activation and of the responses generated along this membrane results in a conformational change, which enables signaling cascade with a particular emphasis placed on AML. the activation loop of the kinase to be phosphorylated on Thr 308 by phosphoinositide-dependent kinase-1 (PDK-1, which also requires 3-phosphorylated inositol lipids for activa- PI3K family of isozymes and their activation tion and plasma membrane translocation) and at Ser 473 in the C-terminal hydrophobic motif by a kinase (often referred The large family of PI3K lipid in mammalian cells has to as PDK-2) whose identity, despite intense investigation, been categorized into three classes, referred to as I, II and III, remains highly controversial.15,16 Candidate PDK-2s include each of which has its own characteristics in terms of molecular integrin-linked kinase, DNA-dependent and structure and substrate specificity.26 Class I PI3Ks are the best mitogen-activated protein kinase-kinase 2. The corresponding understood and are key players of multiple intracellular sites of Akt2 are Thr 309 and Ser 474, whereas signaling networks that integrate a wide variety of signals, those of Akt3 are Thr 305 and Ser 472.15 Recent evidence has engaged by many polypeptide growth factors. For this reason, highlighted that Ser 473 phosphorylation precedes Thr 308 they will be the only isoforms considered relevant to this review. phosphorylation and actually enhances it (see Sarbassov et al.36 Growth factor receptors drive activation of class I PI3Ks either and references therein). directly or via associated tyrosine kinases, heterotrimeric G Furthermore, it has been shown that additional phosphory- proteins or Ras. Class I PI3K preferred in vivo substrate is lative steps may increase Akt activation, including phosphory- 15 phosphatidylinositol 4,5 bisphosphate (PtdIns (4,5)P2), which is lation of multiple tyrosine residues, and phosphorylation on phosphorylated to yield phosphatidylinositol 3,4,5 trisphosphate Ser 129 through (CK2),37 but relevance of these (PtdIns (3,4,5)P3). They are further divided into class IA and IB phosphorylative events awaits full elucidation. Phosphorylated PI3Ks. Class IA PI3Ks are composed of heterodimers of an Akt migrates to both the cytosol and the nucleus. Nuclear Akt adaptor/regulatory (either p85 or p55) and a p110 catalytic may fullfil an important antiapoptotic role.38 However, the subunit.27 There are at least seven adaptor/regulatory proteins relative contribution of Akt signaling at the plasma membrane, that are generated by expression and alternative splicing of three the cytosol and the nucleus also remain to be determined. different genes (referred to as Pik3r1, Pik3r2 and Pik3r3), For the scopes of this review, it may be worth mentioning here whereas three p110 isoforms have been identified: a, b and that Akt activity is modulated by a complex network of d.28 These are encoded by three different genes, PI3KCA, regulatory proteins that interact with the PH domain, or the PI3KCB and PI3KCD. The adaptor/regulatory subunits act to kinase domain or the C-terminal of Akt.39 One of these proteins localize PI3K to the plasma membrane by the interaction of their is heat-shock protein-90 (HSP-90), a molecular chaperone that Src homology 2 (SH2) domains with phosphotyrosine residues in forms a complex with the co-chaperone Cdc37. This complex activated receptors. They also serve to stabilize p110 and to binds a variety of proteins, including tyrosine and serine/ limit its activity. Insulin and some growth factors preferentially threonine protein kinases.40 The HSP-90/Cdc37 complex inter- signal through p110b.29 acts with the Akt kinase domain. Therefore, small molecules The single class IB PI3K or PI3Kg is made of a p110g catalytic capable of disrupting such an interaction may represent valid subunit and a p101 regulatory subunit, unrelated to p85. p110g drugs to block Akt function. signals downstream of heterotrimeric G proteins and Ras26 and It should also be emphasized that PI3K/Akt signaling can be its upregulation is a hallmark of inflammation.30 upregulated by many forms of cellular stress including heat shock, low pH, ultraviolet light, , hypoxia, hypoglyce- mia and oxidative stress.41 Stress-induced PI3K/Akt upregulation Akt isoforms and their activation is to be viewed as a compensatory protective mechanism which cells activate for escaping death. This is very relevant to the Akt, a serine/threonine protein kinase also known as protein topic of this review, because neoplastic cells perceive chemo- kinase B (PKB), is the mammalian homolog of the transforming therapy as an insult, and many types of chemotherapy exert viral oncogene v-Akt that causes murine T-cell lymphoma.31 their cytotoxic effects through the generation of reactive oxygen Akt, which belongs to the AGC kinase superfamily, is now species.42 Consistently, it has been reported that daunorubicin known to include three closely related, highly conserved rapidly upregulated the PI3K/Akt pathway in U937 human isoforms encoded by the following distinct genetic loci: Akt1/ leukemia cells.43 The exact molecular mechanism underlying a, Akt2/b and Akt3/g.15,16 Akt1 is ubiquitously expressed at high this activation is unclear, even though it is now well established levels with the exception of the kidney, liver and spleen. Akt2 that apoptogenic stimuli quite often initiate an antagonistic expression varies between different organs, with higher expres- antiapoptotic program.44,45 sion levels in the skeletal muscle, intestinal organs and reproductive tissues. Akt3 is not detected in several tissues where Akt1 and Akt2 are abundantly expressed, but it is Negative regulation of the PI3K/Akt pathway relatively highly expressed in the brain and testis.31,32 knockout studies have defined the biological importance of Akt As 3-phosphorylated inositides are not hydrolyzed by any isoforms in normal cells. In particular, Akt2-null mice develop a known , a counter-regulation by phosphatases typical type II diabetes,33 whereas Akt1- and Akt3-deficient has emerged as a crucial process to control PI3K-dependent mice are not diabetic but display a decrease in size of all the signaling. PTEN (Phosphatase and TENsin homolog deleted on

Leukemia PI3K/Akt signaling in AML AM Martelli et al 913 10) is a dual specificity lipid and protein expressed as a membrane-bound or soluble form by bone phosphatase that preferentially removes the 3-phosphate mainly marrow stroma cells. It has been disclosed that FL–FLT3 from PtdIns (3,4,5)P3 but is also active on PtdIns (3,4,)P2, thereby interaction plays an important role in the proliferation and antagonizing PI3K/Akt signaling network.46 PTEN-inactivating differentiation of hematopoietic cells. FLT3 is also expressed in a mutations or silencing occur in a wide variety of human cancers high proportion of AML cells. Activating mutations of FLT3 are (including glioblastoma, melanoma, prostatic and endometrium the second most frequent genetic lesions in AML (nucleophos- carcinomas) and this results in Akt upregulation.47 Therefore, min mutations are now considered to be the most common, see PTEN is a tumor suppressor acting upstream of Akt.48 Two other Noguera et al.63), and AML patients with FLT3 mutations have a phosphatases, SHIP-1 and SHIP-2 (for SH domain-containing worse prognosis than those with normal FLT3.64 In addition, inositol phosphatases), are capable of removing the 5-phosphate mutations of the FLT3 tyrosine kinase domain (FLT3-TKD) 49 from PtdIns (3,4,5)P3 to yield PtdIns (3,4,)P2. Whereas SHIP-1 activation loop have been detected in a minority (7%) of is predominantly expressed in hematopoietic cells, SHIP-2 is patients with AML. However, their functional consequences more ubiquitous. However, their role on Akt function is not well are not well understood and so far they have not been shown understood, and in some cases they could not reverse Akt to be of significant prognostic relevance.65 Moreover, in a activation, something PTEN can do.49 2A mouse model of myeloid precursors, FLT3-TKD activation (PP2A), which is rapidly emerging as a new oncosuppressor,50 is loop mutations were not associated with increased PI3K/Akt capable of directly dephosphorylating and downregulating activity.61 Akt,51,52 whereas recent work indicates that Ser 473 phospho- About 80% of AML patients have blast cells that express c-Kit, Akt is dephosphorylated by a PP2C family phosphatase, referred another class III receptor tyrosine kinase for the stem cell factor to as PHLPP, another candidate tumor suppressor.53 (SCF) ligand.66 Mutations in the extracellular or intracellular portions of c-Kit are detected in approximately 20–30% of AML patients with t(8;21) or inv(16)/t(16;16).65 These mutations are Activation of the PI3K/Akt signaling network in AML known for activating PI3K/Akt; however, these results have not been obtained with AML cell lines and/or blasts.67,68 A gain-of- Recently, several papers have highlighted that constitutive function point mutation of c-Kit (Asn822Lys) has been reported activation of PI3K/Akt signaling is a common feature of in the Kasumi-1 AML cell line, which carries the t(8;21) AML.21–24,54–57 From 50 to 70% of patients with AML display translocation. PI3K-dependent activation of Akt was observed phosphorylation of both Thr 308 and Ser 473 Akt. No in Kasumi-1 cells.69,70 Therefore, the outcome of c-Kit mutations correlation was shown to exist between Akt phosphorylation on PI3K/Akt signaling needs to be further explored in AML cell levels and French–American–British (FAB) subtype of AML, lines and blasts. percentage blast infiltration of the bone marrow, cytogenetic Moreover, it has been recently demonstrated that the PI3K anomalies, or when comparing untreated versus relapsed/ p110d catalytic subunit isoform was consistently expressed at refractory AML.54 However, the overall survival time for patients high levels in AML blasts, in contrast to the other class I isoforms demonstrating Akt activation was significantly shorter when (a, b), of which the expression was very variable among patients, compared to patients with no Akt activation.22 Although the even if, in some of them, was quite high. Interestingly, IC87114, mechanisms that upregulate PI3K/Akt signaling in AML cells a p110d-selective pharmacological inhibitor, suppressed both remain unclear, Akt activation in AML blasts may be dependent constitutive and FL-stimulated Akt activation in AML blasts to on, or independent from, PI3K.21 the same extent as LY294002, a non-selective inhibitor of PI3K In about 15–25% of AML cases, N-Ras or K-Ras gene point isozymes.71 However, the reason for higher expression of p110d mutations have been detected. These mutations abrogate Ras in AML blasts remains unclear. Originally, this isoform had been intrinsic GTPase activity and lead to constitutive Ras activation thought to be specific for cells of hematopoietic lineage, but with a consequent stimulatory effect on the PI3K/Akt pathway.58 more recently it has also been detected in some non- Indeed, it is well established that Ras can activate the PI3K/Akt hematopoietic cell types, especially those of breast or melano- axis either by itself or through the Raf/MEK/ERK pathway.46 cytic origin, both in the untransformed and transformed state.72 Up to 20–25% of AML patients harbor internal tandem Conceivably, the above findings are not mutually exclusive, duplication (ITD) of the juxtamembraen domain of FLT3. This because activated Ras, FLT3 or c-Kit could impinge on elevated mutation results in ligand-independent dimerization of FLT3 and levels of p110d, also considering that upregulation of PI3K constitutive upregulation of its tyrosine kinase activity, ensuing p110d activity in AML blasts does not seem to be dependent in stimulation of downstream signaling pathways, including on activating mutations.73 No mutations have been found in PI3K/Akt.59 Surprisingly, however, most of the papers focusing the gene coding for p110a in AML blasts,74 whereas PI3KCA on FLT3-ITD and PI3K/Akt upregulation show data obtained is frequently amplified or harbors activating mutations in with mouse cells such as 32D or BaF3.60–62 Importance of FLT3- several solid tumors, including gastric, colon, breast and liver ITD in causing PI3K/Akt upregulation of mouse myeloid cancer.75,76 precursors is demonstrated by a study in which overexpression As to PTEN, a recent study highlighted that PTEN phosphory- of FLT3-ITD cDNA resulted in constitutive activation of Akt, lation was present in approximately 75% of AML patients. which phosphorylated and inhibited the transcription factor PTEN phosphorylation was significantly associated with Akt FoxO3 (see later). Restored FoxO3 activity reversed FLT3-ITD- phosphorylation and with shorter overall survival.77 It is known mediated growth properties and dominant-negative Akt pre- that phosphorylation at the C-terminal regulatory domain of vented FLT3-ITD-mediated independence of 32D PTEN stabilizes the molecule, but makes it less active towards its 54 78 myeloid precursors. Overall, there is need for investigations substrate, PtdIns (3,4,5)P3. Moreover, PTEN expression has showing that in AML blasts FLT3-ITD directly correlates with been shown to be low or absent in some AML patients,23 PI3K/Akt upregulation. although the level of PTEN expression did not always correlate It is worth remembering here that FLT3, a member of the class with the degree of Akt phosphorylation. However, a subsequent III receptor tyrosine kinases, is preferentially localized on the study failed to demonstrate that AML blasts have a decreased cell surface of hematopoietic progenitors, and its ligand (FL) is expression of PTEN.55

Leukemia PI3K/Akt signaling in AML AM Martelli et al 914 A study of 62 AML patients, showed that 15 of them had has been most intensely investigated. It is now clear that Akt aberrant PTEN transcripts. However, all the samples with enhances survival by directly phosphorylating key regulatory abnormal transcripts also displayed normal full-length tran- proteins of the apoptotic cascades. Akt phosphorylates Bad, a scripts, suggesting that aberrant transcripts could result from proapoptotic member of the Bcl-2 family, at Ser 136. This altered RNA splicing. Morover, no loss of heterozygosity or phosphorylation event promotes Bad sequestration by 14-3-3 mutations were found.79 proteins in the cytosol, thereby preventing Bad from interacting As far as PTEN-inactivating mutations are concerned, they do not with either Bcl-2 or Bcl-XL at the mitochondrial membrane. The seem to occur very frequently in AML.80,81 Therefore, the role, if final effect is inhibition of apoptosis.95 Treatment with the PI3K any, of PTEN in causing Akt activation in AML blast cells is unclear. inhibitor LY294002 reduced Ser 136 Bad phosphorylation and As to other lipid or protein phosphatases, a study has induced apoptosis of AML blasts with constitutively active PI3K/ implicated a dominant-negative SHIP-1 mutation as a possible Akt pathway.24 This finding is an indication of the key role cause of Akt activation in AML.82 Low levels of PP2A have also played by phosphorylated Bad to prevent apoptosis of AML been reported in some AML types, but correlation with Akt cells. PI3K/Akt-dependent Bad phosphorylation has also been activation is lacking.83 detected in HL60 leukemia cells.88 A similar negative regulation Other possible activation mechanisms of the PI3K/Akt has been demonstrated for Yes-associated protein, whose cascade in AML cells have been recently proposed. Vascular phosphorylation by Akt leads to repression of p53-related endothelial growth factor (VEGF) is a powerful angiogenic transcription factor p73 and reduced expression of the molecule for hematological malignancies. It behaves as a proapoptotic protein Bax.96 Conflicting results exist in the critical regulator of endothelial cell survival, motility and literature as to p73 expression in AML blasts, because in some proliferation.84 It is intriguing that AML blasts synthesize and studies it has been reported that the p73 gene was not expressed secrete VEGF and have demonstrable VEGF receptors, that is, owing to promoter hypermethylation,97 whereas in others p73 VEGFR-1 and VEGFR-2.85 Using KG1 and HL60 human protein was detected in most of the investigated cases.98 Also, leukemic cell lines as experimental models, it has been shown Bax expression in AML is a controversial issue, with some that VEGF elicited a rapid and sustained Akt phosphorylation investigations reporting high levels and others demonstrating through a mechanism which was dependent on PI3K because it low levels.99,100 Therefore, it is difficult at present to draw a firm could be inhibited by wortmannin.86 In addition, in AML blasts conclusion about the relationship (if any) between PI3K/Akt and human acute leukemia cell lines, angiopoietins activated activation and p73/Bax expression in AML blasts. PI3K through an autocrine mechanism.87 Therefore, at least in Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/ some AML cases, upregulation of the PI3K/Akt axis might be due JNK) is an important mediator of apoptosis in cells exposed to a to an autocrine and/or paracrine production of angiogenic variety of noxious stimuli, including chemotherapeutic drugs.101 factors, such as VEGF and angiopoietins. Akt may interfere with SAPK/JNK signaling and inhibit apoptosis Our laboratory has shown that in a HL60 cell line subclone, by phosphorylating and thereby inactivating ASK1, a protein activation of PI3K/Akt signaling was dependent on autocrine kinase which transduces signals to SAPK/JNK.16 In U937 secretion of insulin-like growth factor-1 (IGF-1).88 At present, leukemia cells, Akt signaling negatively regulated SAPK/ there is no evidence that a similar mechanism could be effective JNK,102 at variance with previous results obtained with HL60 in AML blasts, even though it is known that the growth of these cells.103 Available evidence suggests that SAPK/JNK is not cells is increased in vitro in response to IGF-1 stimulation.89 activated in AML blasts under basal conditions, but we could Nevertheless, we feel that this possibility should be further not exclude, it does in response to noxious stimuli such as explored, because over the past 5 years multiple large case– chemotherapeutic drugs.104 control studies have reported positive associations between high In another mechanism to thwart apoptosis, Akt promotes circulating levels of IGF-1 and risk for different types of phosphorylation and nuclear translocation of Mdm2, an E3 cancer.90 Regarding malignant hematopoietic disorders, the which mediates ubiquitinylation and protea- role of IGF-1 in promoting proliferation, survival and drug some-dependent degradation of the p53 tumor suppressor resistance of multiple myeloma cells through PI3K/Akt signaling protein,105,106 thereby downregulating p53 and antagonizing is well established.91 p53-mediated cell cycle checkpoint. Consistently, blockade of Finally, interactions between very late antigen (VLA)-4 (a4b1 integrin) on leukemic cells and fibronectin on bone marrow cells has been shown to activate PI3K/Akt signal transduction network in U937 and HL60 human leukemia cell lines.92 However, some Table 1 Possible causes for PI3K/Akt activation in AML cell lines and/or blasts aspects of this have been contested by another group.93 In Table 1 and Figure 1, we summarize the possible mecha- nisms of activation of PI3K/Akt signaling in AML cells. Cause Cell type References Whatever the reason might be for PI3K/Akt activation, it is Activating Ras mutations AML blasts 58 very important to emphasize that recent results have highlighted Activating c-Kit mutations Kasumi-1 69,70 that upregulation of PI3K/Ak axis is present not only in the bulk Upregulation of PI3K p110d AML blasts 71 77 of the AML cell population, but also in LSCs tranplanted in PTEN phosphorylation AML blasts 23 NOD/SCID mice,94 where it exerts a powerful prosurvival PTEN downregulation AML blasts Inactivating SHIP-1 mutation AML blasts 82 effect. This finding indicates that therapeutical targeting of the Autocrine VEGF secretion KG1, HL60; AML blasts 86 PI3K/Akt pathway has the potential for eradicating AML. Autocrine angiopoietin secretion KG1, HL60; AML blasts 87 Autocrine IGF-1 secretion HL60 88 VLA-4/fibronectin interaction U937, HL60 92 Antiapoptotic targets of PI3K/Akt pathway Abbreviations: AML, acute myeloid leukemia; IGF-1, insulin-like growth factor-1; PI3K, phosphoinositide 3-kinase; PTEN, phosphatase and Because Akt is the prototypic kinase which promotes cellular TENsin homolog deleted on chromosome 10; VEGF, vascular survival to apoptotic insults, survival by Akt is the process that endothelial growth factor.

Leukemia PI3K/Akt signaling in AML AM Martelli et al 915

Figure 1 Possible mechanisms leading to PI3K/Akt signaling upregulation in AML cell lines and/or AML blasts. In this simplified cartoon, mutated FLT3 or Ras, and growth factors (vascular endothelial growth factor , insulin-like growth factor-1) impinge upon elevated levels of PI3K p110d. Other possible reasons for enhanced PI3K/Akt include hyperphosphorylation of PTEN and inactivating SHIP mutation which could result in high levels of PtdIns (3, 4, 5)P3. Cytosolic inactive Akt (Akt off) is recruited to the plasma membrane where it is activated (Akt on) by phosphorylation on Thr 308 (by PDK-1) and Ser 473 (by the putative PDK-2). Active Akt targets a series of substrates which are fundamental for cell proliferation, survival and translation. Arrows indicate activating phosphorylation events, whereas perpendicular lines indicate inhibitory events. RTK, receptor tyrosine kinase. the PI3K/Akt axis led to a more than twofold increase in p53 In addition to downregulating FoxO activity, Akt is capable of activity in four out of nine AML patients tested.55 This indicates upregulating nuclear factor-kappa B (NF-kB), a transcription that in some AML cases, p53 is regulated through PI3K/Akt- factor which is deeply involved in the regulation of cell dependent signaling and this pathway could be a mechanism to proliferation, apoptosis and survival.115,116 The survival-promot- promote resistance to cytotoxic agents.107 ing activity of NF-kB is mediated by its ability to induce Moreover, Akt may promote cell survival by phosphorylating expression of antiapoptotic proteins including cIAP-1 and -2, transcription factors that control the expression of pro- and anti- XIAP, c-FLIP and TRAFs, which oppose caspase activation. apoptotic genes. Akt either negatively affects factors that Members of the NF-kB family form dimers (classically hetero- promote death or positively regulates factors dimers of p65 with p50) that, under non-stimulated conditions, inducing survival genes. An example of the former is the FoxO are retained in the cytoplasm. NF-kB function is regulated family of transcription factors, previously referred to as forkhead through its association with the inhibitory I-kB, which transcription factors.108 Phosphorylation of FoxO factors by Akt sequesters NF-kB. Phosphorylation of I-kB by upstream kinases, alters their intracellular localization: in the absence of Akt referred to as IKKs, promotes its degradation via the ubiquitin- activation, FoxO proteins are predominantly localized in the pathway. This, in turn, allows NF-kB nuclear nucleus where they are able to promote transcription of translocation and upregulation of target genes.117 Akt phosphor- proapoptotic target genes such as Fas ligand (Fas-L) and Bim. ylates directly and activates IKKa and, more importantly, it is Activation of the PI3K/Akt pathway leads to nuclear export of believed to be essential for IKK-mediated destruction of I-kB.16 these transcription factors. In the nucleus, phospho-FoxO factors Our laboratory has shown that the PI3K/Akt axis regulates specifically interact with 14-3-3 proteins, which serve as NF-kB-dependent gene expression in HL60 cells.118,119 An in- chaperone molecules to escort them out of the nucleus. Once creased level of activated nuclear NF-kB has also been reported in the cytoplasm, they are degraded via the ubiquitin-protea- in myeloid blasts120,121 which is frequently mediated by the some pathway.109,110 FoxO factor phosphorylation requires PI3K/Akt signaling network.55,58 intranuclear localization of active Akt, which has been All in all, the importance of the PI3K/Akt pathway in documented in both HL60 cells111 and AML blasts.54 Akt determining survival of AML blasts is emphasized by the phosphorylation was found to be significantly associated with observation that a pharmacological inhibitor of this network elevated levels of phospho-FoxO1 and phospho-FoxO3 (pre- (LY294002), when employed alone, greatly enhanced the viously referred to as FKHR and FKHRL1, respectively) factors in apoptosis rate of AML cells in culture.23,24 AML blast cells.55,112 Patients with phospho-FoxO1 had a significantly shorter overall survival than those without,112 as it could be expected for patients with upregulated PI3K/Akt axis. PI3K/Akt and cell cycle regulation Although there is no information about the levels of Bim in AML patients, Fas-L has been found to be consistently upregulated in Akt targets p27Kip1, a direct inhibitor of -dependent AML blasts.113,114 This may indicate that in AML cells kinase (cdk) 2, one of the cdks responsible for the activation of expression of this proapoptotic protein is not regulated through E2F1 transcription factors that promote DNA replication.122 a PI3K/Akt/FoxO-dependent pathway. When phosphorylated by Akt on Thr 157, p27Kip1 mainly

Leukemia PI3K/Akt signaling in AML AM Martelli et al 916 localizes to the cytoplasm where it cannot exert its inhibitory effect, so that cell proliferation is enhanced.16 A direct relationship between cytoplasmic localization of p27Kip1 and PI3K/Akt activation has been demonstrated in HL60 cells.111 Interestingly, cytoplasmic localization of p27Kip1 in AML blasts with upregulated Akt activity was significantly associated with shorter disease-free and overall survival.123,124 Cyclin D1 levels were also found to be upregulated through PI3K/Akt signaling in HL60 cells.111 This might depend on Akt-mediated inhibition of glycogen synthase kinase 3b (GSK3b) (see later), because cyclin D1 phosphorylation by GSK3b results in its destabilization.125 However, enhanced cell proliferation could also be a con- sequence of nuclear exclusion of FoxO factors, because these transcription factors, once in the nucleus, upregulate expression of three target genes which lead to G1/S arrest, p27Kip1, p21Waf/Cip1 and the retinoblastoma family member p130.126–128 Moreover, FoxO factors can also promote cell cycle arrest by repressing the expression of cyclin D1 and D2, two positive cell cycle regulators.129,130 It should be pointed out, however, that a recent investigation failed to unveil any relationship between Figure 2 The Akt/mTOR pathway. Active Akt inhibits TSC2 activity through direct phosphorylation. Tuberous sclerosis 2 is a GTPase- p27Kip and p21Waf/Cip1 expression and activation of PI3K/Akt 55 activating protein that functions in association with the putative TSC1 signaling in AML blasts. Therefore, how activation of the PI3K/ to inactivate the small Rheb. Akt-driven TSC1/TSC2 Akt axis could positively influence proliferation of AML cells complex inactivation allows Rheb to accumulate in a GTP-bound remains to be fully elucitated. state. Rheb-GTP then activates through a mechanism not yet elucidated the protein kinase activity of mTOR when complexed with the Raptor adaptor protein and mLST8. Moreover, Akt can directly phosphorylate and activate mTOR. Mammalian target of rapamycin PI3K/Akt and metabolism downstream targets include p70S6K and 4E-BP1. Once phosphory- lated by mTOR, 4E-BP1 dissociates from eIF-4E which can then initiate The mammalian target of rapamycin (mTOR) is a serine/ translation. However, mTOR also exists complexed with Rictor/ threonine kinase which regulates translation in response to mLST8. This complex phosphorylates Akt on Ser 473 and could be nutrients/growth factors by phosphorylating components of the phosphoinositide-dependent protein kinase-2. Rapamycin binds the protein synthesis machinery, including p70S6 kinase (p70S6K, a immunophilin FK506-binding protein 12 and then inactivates the mTOR/Raptor/mLSTB complex, but not the m/TOR/Rictor/mLST8 ribosomal kinase) and eukaryotic initiation factor (eIF)-4E complex. Arrows indicate activating events, whereas perpendicular binding protein (4EBP)-1, the latter resulting in release of the lines indicate inhibitory events. translation initiation factor eIF-4E, allowing eIF-4E to participate in assembly of a translational initiation complex.131 Conceiva- bly, mTOR acts as a checkpoint sensor indicating to cells that (rapamycin-insensitive companion of mTOR)/mLST8 complex, there are sufficient nutrients available to proceed through the which displays rapamycin-insensitive activity. It should also be cell cycle.132,133 Therefore, mTOR regulates a variety of steps reminded here that Akt directly phosphorylates and activates involved in protein synthesis, but in particular favors the mTOR, and this is to date the only example of Akt-mediated production of key molecules such as c-Myc, cyclin D1 and phosphorylation which results in substrate activation.139 mTOR ribosomal proteins.134 p70S6K, which can also be directly was found to be phosphorylated in AML blasts, along with activated by PDK-1, phosphorylates the 40s ribosomal protein, its two downstream substrates, p70S6K and 4EBP-1, in a PI3K/ S6, leading to active translation of mRNAs.135 By controlling Akt-dependent fashion.23 Nevertheless, others failed to detect protein synthesis, p70S6K and 4E-BP1 also regulate cell growth any relationship between PI3K/Akt upregulation and p70S6K and hypertrophy, which are important processes for neoplastic phosphorylation in AML primary cells.55 progression. Therefore, even more distal steps in the PI3K/Akt Another Akt substrate important for metabolic function is pathway may have the potential to be exploited for cancer GSK3b, which phosphorylates and inactivates glycogen treatment. Akt-mediated regulation of mTOR activity is a synthase in response to insulin stimulation. When phosphory- complex multistep phenomenon (Figure 2). Akt inhibits tuberous lated by Akt on Ser 9, GSK3b is downregulated.140 Glycogen sclerosis 2 (TSC2 or hamartin) function through direct phos- synthase kinase 3b is phosphorylated in AML cells with phorylation.136 Tuberous sclerosis 2 is a GTPase-activating upregulated Akt function.77 However, as for p70S6K, others protein (GAP) that functions in association with the putative found that downregulation of PI3K/Akt signaling in AML primary TSC1 (or tuberin) to inactivate the small G protein Rheb (Ras cells did not result in GSK3b dephosphorylation.55 Thus, further homolog enriched in brain).137 Tuberous sclerosis 2 phosphor- investigations are necessary to elucidate the role played by PI3K/ ylation by Akt represses GAP activity of the TSC1/TSC2 Akt in controlling p70S6K and GSK3b function in AML blasts. complex, allowing Rheb to accumulate in a GTP-bound state. Interestingly, GSK3b has been implicated in the signaling Rheb-GTP then activates through a mechanism not yet pathway elicited by Wnt, a ligand for transmembrane receptor elucidated the protein kinase activity of mTOR when complexed frizzled. The b-catenin protein is at the core of the canonical with the Raptor (regulatory-associated protein of mTOR) adaptor Wnt signaling pathway. Wnt stimulation leads to b-catenin protein and mLST8.138 The mTOR/Raptor/mLST8 complex is accumulation, nuclear translocation and interaction with sensitive to rapamycin and, importantly, inhibits Akt via a transcription factors to regulate genes important for embryonic negative feedback loop which involves, at least in development and proliferation.141 The Wnt/b-catenin signaling part, p70S6K.138 The relationship between Akt and mTOR is network is constitutively activated in AML blasts as a result of further complicated by the existence of the mTOR/Rictor the expression of transcription factor fusion proteins such as

Leukemia PI3K/Akt signaling in AML AM Martelli et al 917 Table 2 PI3K/Akt downstream targets identified in AML cell lines whereas forced expression of constitutively active Akt rendered and/or blasts HL60PT cells less sensitive to chemotherapeutic drugs or ATRA.88 Importantly, involvement of PI3K/Akt axis in chemo- Target Cell type References resistance has been demonstrated in AML primary cells.55 The PI3K/Akt/NF-kB module is also responsible for human Bad HL60; AML blasts 24,88 71,102 leukemia cell line and APL blast resistance to arsenic trioxide SAPK/JNK U937; AML blasts 119,147,148 149 Mdm2/p53 AML blasts 55 (As2O3), another effective agent for APL treatment. FoxO transcription HL60; AML blasts 54,55,111,112 It is interesting that PI3K/Akt signaling inhibitors decreased the factors intracellular glutathione content, and caused intracellular IKK/I-kB/NF-kB and HL60; AML blasts 55,58,118–121,158, oxidation, as revealed by peroxide accumulation measurement. 219,225,226 related antiapoptotic Cotreatment with subcytotoxic concentrations of hydrogen genes 208 peroxide increased apoptosis induction by As O . On the other Mcl-1 HL60; NB4 2 3 p27Kip1 HL60; U937; AML blasts 111,123,133 hand, the treatments did not significantly affect glutathione Cyclin D1 HL60 111 S- p expression and activity. These results, which mTOR/p70S6K/4EBP-1 AML blasts 23,84,204 highlighted glutathione as a novel target of PI3K/Akt in myeloid GSK3b AML blasts 77 leukemia cells, may partially explain the selective increase of 147 Abbreviations: AML, acute myeloid leukemia; GSK3b, glycogen As2O3 toxicity by PI3K/Akt inhibitors, even though this may synthase kinase 3b;I-kB, inhibitory cofactor kappa B; mTOR, also be in relationship with reduced expression of antiapoptotic mammalian target of rapamycin; NF-kB, nuclear factor kappa B; proteins that counteract As2O3-dependent caspase activation, as PI3K, phosphoinositide 3-kinase; SAPK/JNK, stress-activated protein shown by our group.119 kinase/c-Jun N-terminal kinase. Moreover, PI3K/Akt pathway is involved in the resistance to tumor necrosis factor-related apoptosis inducing ligand (TRAIL).118 TRAIL is one of the members of the tumor necrosis AML1/ETO142,143 and there is evidence for neoplastic myeloid factor superfamily known to induce apoptosis in a wide variety transformation supported by this pathway.144 Phosphorylation of cancer, but not normal, cells.150,151 The use of TRAIL or of of b-catenin by GSK3b on key N-terminal residues targets it for agonistic to TRAIL receptors as therapeutic agents for ubiquitination and breakdown in the proteasome.143 Given that AML has been proposed.151 A main problem, emerging from Akt phosphorylates and inactivates GSK3b, upregulation of recent in vitro studies, is that AML blasts express low levels of PI3K/Akt signaling in AML cells might increase the levels of TRAIL receptors and are therefore intrinsically resistant to this b-catenin resulting in its accumulation and translocation to the molecule.152,153 However, expression of TRAIL receptors could nucleus where it would stimulate the transcription of target be increased by cotreating AML cells with a histone deacetylase genes that include c-Myc and cyclin D1. This may well be an inhibitor.154 Our results have clearly established that PI3K/Akt/ example of interdependence between alterations in signaling NF-kB upregulation resulted in enhanced expression of the pathways and changes in transcriptional activity of AML blasts, FLICE inhibitory protein, cFLIP(L), in TRAIL-resistant HL60AR as outlined above. cells which, despite the expression of TRAIL receptors, did not Downstream targets of PI3K/Akt pathway identified in AML undergo apoptosis when exposed to TRAIL.118 cFLIP(L) is an cell lines or blasts are summarized in Table 2. inhibitor of caspase-8, the apical caspase of the death signaling cascade elicited by TRAIL.155 PI3K pharmacological inhibitors restored the sensitivity of HL60AR cells to TRAIL along with a PI3K/Akt activation and AML resistance to therapeutic downregulation of cFLIP(L) expression levels.118 Even though treatments there is at present no clear information about upregulation of cFLIP(L) in AML cells, it may be envisaged that a combined The largely acknowledged, fundamental role played by PI3K/Akt treatment consisting of a histone acetylase inhibitor and a PI3K/ cascade in opposing apoptosis has led to an intense investiga- Akt inhibitor, would overcome TRAIL resistance of AML blasts tion into contribution of this signaling pathway to tumor cell owing to the lack of TRAIL receptor expression concomitantly survival in response to various types of therapeutic treatment. In with elevated cFLIP(L) levels. In this connection, it is worth a wide variety of neoplastic cells, PI3K/Akt signaling is deeply recalling here that the amount of XIAP, another member of involved in resistance to classical antineoplastic chemother- inhibitors of apoptosis which is under the control apeutic agents including etoposide, anthracyclins and cispla- of NF-kB, is strongly related to prognosis in AML patients, tin.41 The importance of the PI3K/Akt network in causing because patients with lower levels of XIAP protein had resistance to drugs commonly used for AML treatment was first significantly longer survival and a tendency toward longer demonstrated by O’Gorman et al.145 These authors employed remission duration than those with higher levels of XIAP.156 drug-resistant HL60 human leukemia cells to show that either However, these conclusions have been challenged by another wortmannin or LY294002 downregulated Akt activity and study.157 Whatever the case, in AML cell lines XIAP expression increased sensitivity to etoposide or doxorubicin. More recently, is at least in part under PI3K control.158 our group has confirmed these findings by taking advantage of a Finally, a recent study has demonstrated that both 32D cells HL60 cell clone, referred to as HL60AR (apoptosis resistant carrying FLT-ITD and AML blasts require concurrent incubation cells) which, when compared with parental (PT) HL60 cells, with rapamycin (an mTOR inhibitor, see later) to undergo displayed a constitutively active PI3K/Akt axis.88 HL60AR cells apoptosis in response to ATRA and the histone deacetylase are much more resistant than HL60PT cells to a wide variety of inhibitor valproic acid cotreatment.159 chemotherapeutic drugs as well as to all-trans retinoic acid Taken together, the aforementioned findings have provided (ATRA), a powerful differentiating agent for HL60 cells that is the rationale for using pharmacological inhibitors of the PI3K/ successfully employed to treat acute promyelocytic leukemia Akt network to overcome resistance to therapeutic strategies that (APL).146 HL60AR resistance to drugs and ATRA could be are currently used (or that might be used in the near future) for lowered by overexpression of dominant-negative PI3K or Akt, the treatment of AML.

Leukemia PI3K/Akt signaling in AML AM Martelli et al 918 Inhibition of the PI3K/Akt pathway to overcome AML However, PI3K inhibitors have a hypothetical advantage in that therapeutic resistance feedback activation of the pathway, which is seen with inhibition of distal components, such as mTOR (see below), The success of tyrosine kinase inhibitors, such as erlotinib, would not be observed. gefitinib, imatinib and the strong rationale to target the PI3K/Akt pathway, as outlined above, has fed optimism that inhibitors of this signal transduction network might have clinical use for Selective PI3K p110 catalytic subunit inhibitors cancer patients. As activation of the PI3K/Akt axis confers As the PI3K catalytic domain is highly conserved among PI3K therapeutic resistance, compounds that inhibit this pathway by family members,165 it is not surprising that neither wortmannin targeting key regulatory proteins such as PI3K, Akt, mTOR or nor LY294002 discriminate among the different PI3K isozymes. NF-kB have potential for new effective therapies. Either used IC87114 is a potent selective inhibitor of PI3K p110d, with a alone or in combination with existing treatments, inhibitors of IC50 ¼ 0.5 mM and a 450-fold selectivity over the other class I the PI3K/Akt pathway may exploit activation of the PI3K/Akt axis PI3K isoforms.169 IC87114 downregulated Akt phosphorylation within AML cells to induce apoptosis and/or enhance the and almost completely blocked proliferation of AML blast cells efficacy of other forms of treatment. We shall now discuss with elevated levels of PI3K p110d protein.71 IC87114 was as several pharmacological inhibitors that selectively target this effective as LY294002 and did not affect the proliferation of signal transduction cascade. However, FLT3160 and Ras normal hematopoietic precursor cells. These findings suggested inhibitors161 will not be considered here, as they also target that in AML patients, selective pharmacological inhibition of additional survival pathways, such as the Erk1/2 signaling PI3K p110d might offer clinical benefit and be less toxic than network.160,162 For the same reason, we shall also not review inhibiting all PI3K activities, even though PI3K p110d plays a either VEGF163 or IGF-1 inhibitors, including the recently critical role in proliferation and development of the immune described NVP-AEW541.164 system. Indeed, it is well known that mice lacking functional PI3K p110d are viable and fertile, whereas mice lacking PI3K p110a or b are embryonic lethal.26 Non-selective PI3K inhibitors Two classical PI3K inhibitors, wortmannin and LY294002 have been widely used for in vitro and in vivo studies on cancer cell PDK-1 inhibitors lines in which they induce apoptosis and/or increase sensitivity As PDK-1 is the kinase responsible for phosphorylating Akt at to chemotherapeutic drugs and TRAIL.41,165,166 Whereas Thr 308, it plays a very critical role in activation of the pathway. wortmannin is a metabolite antibiotic that was first isolated However, PDK-1 also activates other AGC kinases that regulate from Penicillium wortmanni, LY294002 is a synthetic flavonoid cell proliferation and survival, including (PKC), derivative. Wortmannin irreversibly inhibits PI3K by covalent and p70S6K.170 Hence, compounds that target modification of Lys 802 of the p110 catalytic subunit, whereas PDK-1 will likely inhibit these other kinases. Despite the LY294002 is a reversible inhibitor which competes with ATP for intuitive reasoning that a more selective inhibitor should have the ATP- of PI3K.165 However, neither wortmannin lower toxicity and cause fewer side effects, this off-target activity nor LY294002 are entirely specific for the PI3K/Akt pathway, may be advantageous from a therapeutic standpoint but because wortmannin targets phospholipases C, D and A2, obviously complicates their development as drugs that selec- whereas LY294002 downregulates CK2 activity with similar tively target the PI3K/Akt network, even though the use of potency to PI3K.41 The effect of LY294002 on CK2 appears multitargeted kinase inhibitors, such as sorafenib, has been intriguing in light of CK2-dependent Akt phosphorylation, as advocated to treat cancers that are driven by several metabolic mentioned earlier in this article. There are several studies in abnormabilities.171 The staurosporine derivative UCN-01, a which wortmannin or LY294002 have been employed to drug now in phase II clinical trials, has been shown to potently downregulate in vitro the PI3K/Akt axis of human AML cell inhibit PDK-1 in vitro and in vivo, by forming a complex with lines or blasts. As a consequence of the treatment, cells the kinase domain of PDK-1.172,173 UCN-01 is capable of underwent apoptosis and/or became more sensitive to chemo- interacting synergistically with the farnesyltransferase inhibitor therapeutic drugs or TRAIL.23,24,58,88,118 An extremely L744832, thereby increasing apoptosis of HL60 cells and interesting finding, which has emerged from these investiga- AML blasts.102 Moreover, a recent pilot clinical trial of tions, is that normal hematopoietic progenitors were less cytarabine in combination with UCN-01 in patients with affected by PI3K inhibitors, suggesting a preferential targeting relapsed AML, has shown a decline in Akt kinase activation of leukemic cells.23,24 Although the aforementioned studies which was accompanied by a decrease in checkpoint kinase 1 demonstrated that blocking the PI3K/Akt pathway might be a (Chk1) phosphorylation, an activation of JNK, and reduction in valuable approach to treat AML, there are some intrinsic absolute AML blast counts.174 These findings offer a rationale for disadvantages with these inhibitors. Wortmannin is soluble in the cytotoxic action of this combination therapy for AML organic solvents that may severely limit its use in clinical trails. treatment. Nevertheless, UCN-01 also inhibits PKC-a,-b and -g, Currently, water-soluble wortmannin conjugates are being as well as Chk1, so that its specificity of action remains to be developed to circumvent this issue.167 As to LY294002, it has better defined.175 a very short half-life and is insoluble in aqueous solutions. Relatively few in vivo studies have been conducted to demonstrate its efficacy on the inhibition of growth of cancer Selective Akt inhibitors xenogratfs, but some severe side effects, such as dry and scaly Phosphatidylinositol ether analogues. Most small mole- skin, appeared in treated mice.41 Clearly, the use of non- cular weight kinase inhibitors are ATP mimics. The ATP cleft is selective PI3K inhibitors, such as the recently described strongly conserved between kinases, so ATP-competitive in- wortmannin derivative PX-866,168 is likely to be associated hibitors tend to lack specificity and to affect groups of related with undesirable side effects (e.g. hyperglycemia) because of the kinases. An alternative approach to inhibiting Akt is to target its many important physiological targets of this lipid kinase. PH domain and interfere with PtdIns (3,4,5)P3 binding and

Leukemia PI3K/Akt signaling in AML AM Martelli et al 919 membrane translocation. Phosphatidylinositol ether analogues demonstrated that the drug actually targeted Akt in this (PIAs) have been designed to inhibit this interaction. The experimental system. Nevertheless, potential efficacy of perifo- rationale behind the synthesis of these molecules is that they sine in AML with activated PI3K/Akt signaling, is indicated by cannot be phosphorylated by PI3K on the 3-position of the myo- the results of a recent study in which perifosine induced inositol ring. Indeed, they act as competitors for Akt activation at significant apoptosis in multiple myeloma cell lines and patient the plasma membrane, hence they behave as inhibitors down- multiple myeloma cells, characterized by upregulation of PI3K/ stream of PI3K and PDK-1. As outlined above, a conceptual Akt network.191 Moreover, perifosine augmented dexametha- difference in the development of PIAs to inhibit Akt is that sone, doxorubicin, melphalan and bortezomib-induced multiple targeting the PH domain should minimize the lack of specificity myeloma cell cytotoxicity and demonstrated significant anti- observed with compounds that target the ATP-binding domain tumor activity in a human plasmacytoma mouse model. In this 176 of Akt. Indeed, the Akt isoform PH domains are only about investigation, the perifosine IC50 in vitro effective on neoplastic 177 30% identical to PH domains in other proteins. Importantly, plasma cells (1.5–15 mM) was within the range of plasma PIAs selectively induced apoptosis in several cancer cell lines concentrations achieved in vivo (B16 mM) during a phase I that have high levels of Akt phoshorylation and were only study of perifosine in solid tumors.192 Thus, a phase II trial modestly active in tumor cells displaying low levels of of perifosine on multiple myeloma patients has very recently phosphorylated Akt.178 Moreover, one of these compounds, begun. PX-316, displayed in vivo antitumor activity against human The allosteric Akt kinase inhibitors are particularly interesting, MCF-7 breast cancer and HT-29 colon cancer xenografts in because they can target specifically Akt1, Akt2 or both Akt1 and mice. Both these cell lines have an upregulated PI3K/Akt Akt2. Therefore, at least in theory, the use of isozyme-selective network. Remarkably, PX-316 formulated in 20% hydroxypro- Akt inhibitors might result in less pronounced unpleasant side pyl-b-cyclodextrin for intravenous administration was well effects. For example, as outlined above, studies on knockout tolerated in mice and rats with no hemolysis and no mice have revealed that Akt2 plays a very important role in hematological toxicity.179 insulin-mediated glucose homeostasis, as also demonstrated by In keeping with these findings and by exploiting the the fact that this Akt isoform is the most abundantly expressed in experimental system consisting of HL60AR cells, we demon- glucose-sensitive tissues and organs.193 Therefore, it would be strated that one of these PIAs (1L-6-hydroxymethyl-chiro-inositol very important to define if in AML blasts both Akt1 and Akt2 are 2(R)-2-O-methyl-3-O-octadecylcarbonate) was able to restore upregulated, or if only one of the two isoforms is involved in sensitivity to chemotherapeutic drugs, ATRA and TRAIL.180 conveying survival signals. However, according to the available Furthermore, we have tested two novel PIAs with improved evidence, maximal induction of apoptosis by isozyme-selective metabolic stability and anticancer potential, D-3-deoxy-2-O- Akt inhibitors in several cancer cell lines was achieved with the methyl-myo-inositol 1-((R)-2-methoxy-3-(octadecyloxy)propyl Akt1/Akt2 dual inhibitor.185 These isoform-specific inhibitors hydrogen phosphate and D-2,3-dideoxy-myo-inositol 1-((R)-2- were only modestly active in inducing apoptosis in tumor cells, methoxy-3-(octadecyloxy)propyl hydrogen phosphate (see com- but synergized with chemotherapeutic agents to induce pounds PIA5 and PIA6 in Castillo et al.178) on HL60AR cells. apoptosis. Moreover, these compounds have poor solubility They were able to markedly increase sensitivity of HL60AR cells and pharmacokinetics properties that precluded their evaluation 185 to etoposide or cytarabine at a concentration (5 mM), which was in animal tumor model. Overall, Akt inhibition would be not toxic to human cord blood CD34 þ hematopoietic precursor expected to inhibit most, if not all, of Akt substrates. Because we cells.181 Key issues for the development of lipid-based Akt are far away from the identification of all Akt substrates and inhibitors are oral bioavailability and hemolysis, a side efffect critical substrates can vary with cell type, inhibition of that may determine which of these compounds will eventually individual downstream components of PI3K/Akt pathway may emerge as a front runner.176 miss key players that are involved in Akt-mediated cell proliferation and/or survival. Hence, Akt inhibition, such as PI3K inhibition, may offer greater efficacy, albeit at the expense Other Akt inhibitors. Over the past 3 years, several other of potential greater toxicity. Akt inhibitors have been described, including perifosine (a novel orally bioavailable alkylphospholipid that inhibits Akt phos- phorylation by preventing its membrane localization, possibly HSP-90 inhibitors through interaction with the PH domain, see Kondapaka Heat-shock protein-90 is currently receiving consideration as a et al.182), deguelin (a naturally occurring plant rotenoid, whose potential anticancer drug target. The ability of HSP-90 to mechanism of action is unclear, see Chun et al.183), allosteric stabilize client proteins is inhibited by the benzoquinone Akt kinase inhibitors that are isozyme specific and require the ansamycin antibiotic geldanamycin and its derivatives, that PH domain of Akt,184,185 the indazole-pyridine compound occupy the ATP-binding site on HSP-90 and promote protein A-443654,186 API-2/tricribine187 and a cell-permeable neutraliz- degradation via the proteasome pathway.194 Differences be- ing single-chain to Akt.188 Of all the above inhibitors, tween chaperoning complexes of neoplastic and healthy cells only deguelin and perifosine have been tested on AML cell lines. make HSP-90 an attracting target for anticancer treatment. At 10–100 nM concentration, deguelin downregulated Akt Indeed, in neoplastic cells, HSP-90 forms multimolecular phosphorylation of U937 leukemia cells and markedly in- complexes with both high ATP-ase activity and a high affinity creased their sensitivity to etoposide or cytarabine. A 10 nM for the geldanamycin derivative 17-allylamino-17-demethoxy- concentration of deguelin did not negatively affect the survival geldanamycin (17-AAG), whereas in normal cells it exists as a rate of human cord blood CD34 þ cells. Moreover, deguelin was latent form which displays low ATPase activity and low affinity less toxic than wortmannin on erythropoietin (EPO)- and SCF- for 17-AAG.195 These findings have provided a rationale for the induced erythropoiesis from CD34 þ progenitor cells.189 As to enhanced accumulation and selective cytotoxicity of 17-AAG in perifosine, it has been shown that it synergized with a histone cancer cells.194 Several reports have indicated that HSP-90 acetylase inhibitor to induce a decrease of cell proliferation and inhibitors including 17-AAG, either used alone or in combina- an increase in apoptosis of HL60 cells,190 even if it was not tion with other treatments, selectively induced apoptosis in

Leukemia PI3K/Akt signaling in AML AM Martelli et al 920 leukemic cells harboring activating mutations of FLT3196–198 or A synergism has also been reported for a combination c-Kit.199 Akt dephosphorylation was observed in one of these treatment consisting of rapamycin and UCN-01.205 This combi- studies,196 along with downregulation of other FTL3 down- nation treatment resulted in marked potentiation of apoptosis in stream effectors (STAT5a, Erk1/2). However, as for UCN-01, it U937 cells that was accompanied by a decrease in the levels should be pointed out that 17-AAG, by targeting HSP-90, also of Mcl-1. Mcl-1 protein expression levels are related with inhibits other kinases including Chk1,200 so that it cannot be resistance of human leukemias to a variety of chemotherapeutic considered selective for the PI3K/Akt pathway. agents.206,207 Involvement of PI3K/Akt signaling in the regulation of Mcl-1 expression in human leukemia cells has been shown.208 Another combined treatment which resulted in a synergistic Mammalian target of rapamycin inhibitors cytotoxic effect on HL60 leukemia cells is that constituted of An alternative target to either PI3K or Akt could be represented rapamycin and the cell permeable glycolytic inhibitor 3-bromo- by kinases located downstream of Akt, such as mTOR. 2-oxoproprionate-1-propyl ester.209 The efficacy of this combi- Mammalian target of rapamycin is particularly interesting nation treatment conceivably depends on the fact that in cancer because, besides its important role in cell metabolism, it has cells, which are very hypoxic, the PI3K/Akt/mTOR axis elicits the been recently demonstrated to behave as a critical Akt activator long-known ‘Warburg effect’ where glucose uptake, glycolysis by forming a complex with Rictor.36 The Rictor/mTOR complex and lactate production are accelerated without any change in directly phosphorylated Akt on Ser 473 in vitro and facilitated oxygen consumption.210 Enhanced glycolysis could be related to Thr 308 phoshorylation. Therefore, the Rictor/mTOR complex overexpression of hypoxia-inducible factor (HIF)-1a. Mammalian might be the much sought after PDK-2, which phosphorylates target of rapamycin upregulates HIF-1a, primarily by increasing Akt on Ser 473 in response to growth factor stimulation.201 the rate of HIF-1a protein translation.211 Interestingly, VEGF Mammalian target of rapamycin inhibitors are the most expression is increased by HIF-1a.212 developed class of compounds that target the PI3K/Akt The above results seem to indicate that rapamycin or its pathway and include: rapamycin, CCI-779, RAD001 and the derivative, either alone or in combination, might be very phosphorous-containing derivative AP23573.202 Rapamycin is a promising drugs for the treatment of AML. However, in light of macrocyclic lactone antibiotic, produced by Streptomyces recent findings a caveat is necessary, because it is emerging that hygroscopicus, which potently inhibits the growth of cancer mTOR also mediates suppression of PI3K activation. Indeed, cell lines and induces apoptosis. It does not directly inhibit mTOR phosphorylates insulin receptor substrate-1, resulting in mTOR, but rather binds to its immunophilin, FK506-binding its proteosomal degradation and downregulation of IGF-1- protein 12 (FKBP12). Then, rapamycin/FKBP12 complex binds evoked signaling to PI3K/Akt.213,214 Even if there is no evidence to mTOR complexed with Raptor (Figure 2) and inhibits of the existence of a such a negative feedback loop in AML cells, downstream signaling events.202 Rapamycin has been approved it is clear that mTOR inhibition could result in upregulation of by FDA as an immunosuppressant, and in phase I/II clinical trials growth factor-dependent PI3K/Akt signaling. However, the has shown activity against many types of cancer.139 It has, occurrence of this inhibitory mechanism has been very recently however, two disadvantages: poor solubility and chemical demonstrated in multiple myeloma cells, in which either stability. For this reason, ester analogues of rapamycin with rapamycin or CCI-779 treatment resulted in Akt activation improved aqueous stability and solubility have been synthe- in vivo, suggesting that such feedback also takes place sized.203 CCI-779 has been designed for intravenous injection, in hematopoietic cells.215 Although the role of serum IGF-1 whereas RAD001 is available for oral administration. A phase II in supporting proliferation and survival of myeloma cells both clinical trial with CCI-779 in patients with metastatic renal in vitro and in vivo is well established,216 its involvement in AML carcinoma has already been completed and the drug is now in remains to be demonstrated, but could not be completely ruled phase III clinical trials. RAD001 (which is approved in Europe as out. These findings raise a caution about the indiscriminant use an immunosuppressant agent in solid organ trasplantation) and of rapamycin in cancer therapy. AP23573167 are currently undergoing phase I/II clinical trials as In principle, therefore, therapeutic approaches that simulta- antitumor drugs. neously target both PI3K/Akt and mTOR/Raptor complex, like Rapamycin failed to reverse drug resistance in HL60 cells the combination rapamycin/UCN-01, may ultimately prove with upregulated PI3K/Akt signaling.88,145 In contrast, it has more efficacious. Moreover, another potential weakness of been reported that RAD001 was capable of sensitizing U937 rapamycin is represented by the fact that this drug could be leukemia cells to cytarabine.23 The reason for these conflicting exported from cells through ATP-binding cassette (ABC) type findings is unclear. It might be due to the different cell types and/ transporters that mediate multidrug resistance such as 170-kDa or experimental conditions employed for these investigations, P-glycoprotein,217 and AML cells are known to express several nevertheless it could also be related to rapamycin resistance (see of these transporters.218 Rapamycin extrusion by ABC transpor- later). Rapamycin had only a modest effect on primary AML cell ters could also account for marked differences in the drug survival in liquid culture; however, it markedly impaired AML concentration (range: from less than 1 nM to more than 100 nM) blast clonogenicity while sparing normal hematopoietic pre- necessary to achieve IC50 on AML blast clonogenic assay and cursors. Moreover, it was able to induce a significant and rapid could also explain why some patients did not respond at all to clinical response in vivo in four of nine patients with either rapamycin treatment.204 However, other mechanisms of rapa- refractory/relapsed de novo or secondary AML.204 Accordingly, mycin resistance have been identified.135 another group recently reported that mTOR inhibition with rapamycin led to only a modest decrease in AML blast survival in short-term (2 days) cultures, whereas in long-term (7 days) NF-kB inhibitors cultures the effect was more pronounced. Moreover, rapamycin As underlined above, I-kB/NF-kB is upregulated in AML blasts cytotoxicity in short term cultures could be dramatically leading to the expression of antiapoptotic c-IAP2, whereas increased by co-treatment with etoposide.94 Importantly, etopo- unstimulated human hematopoietic progenitor cells do not side toxicity on CD34 þ cells from healthy donors was not express NF-kB.120,219 Importantly, molecular genetic studies enhanced by addition of rapamycin. using a dominant-negative allele of I-kB demonstrated that

Leukemia PI3K/Akt signaling in AML AM Martelli et al 921 inhibition of NF-kB contributed to apoptotic cell death of malignant disorders. There is no doubt that the activation of the LSCs.220 These observations have provided a rationale for the PI3K/Akt pathway confers resistance to therapeutic treatments of use of NF-kB inhibitors as potential therapeutic agents against various types of cancer both in vivo and in vitro, including AML. AML. There exists an extremely wide variety of natural and This finding has driven the frantic development of compounds synthetic NF-kB inhibitors.221 For example, curcumin, a yellow directed against components in the pathway. Nevertheless, a coloring agent from turmeric (Curcuma longa rhizomes), fundamental issue that still awaits answering is: will inhibition commonly used as a spice, is well documented for its medicinal of this signaling network negatively affect human disorders properties in Indian and Chinese systems of medicine. Several without deleterious side effects, such as perturbations of glucose reports have highlighted that curcumin causes apoptosis in homeostasis? In other words, is there a therapeutic window human leukemia cell lines and downregulates NF-kB activ- when such an ubiquitous and fundamental pathway is targeted? ity.222,223 Capsaicin, a homovanillic acid derivative found in In vitro data show that inhibitors are preferentially cytotoxic for pungent fruits, is another natural NF-kB inhibitor which has tumor cells that exhibit increased PI3K/Akt activation, suggest- been shown to induce growth inhibition and apoptosis of ing that death of cancer cells without death of healthy cells is human myeloid leukemic cell lines in vivo and in vitro.224 Two possible. Perhaps a basis for a potential therapeutic window other natural NF-kB inhibitors have been recently described in vivo can be attributed to increased reliance on pathways and demonstrated to possess in vitro pharmacological activity promoting cellular survival by cancer cells exposed to forms against primary AML cells. Resveratrol, an edible polyphenolic of stress (e.g. chemotherapy) that are known for activating PI3K/ stilbene found in the skin of red grapes and various other fruits, Akt. In addition, tumor cells might be more sensitive than inhibited NF-kB activity of AML cell lines and blasts. NF-kB normal cells to inhibition of this network because they often inhibition correlated with increased apoptosis.225 The other grow in harsh conditions deprived of nutrients and would natural compound capable of downregulating NF-kB in AML therefore be highly reliant on a signaling pathway which has blasts is indole-3-carbinol, which is found in Brassica species been upregulated during disease progression for their survival vegetables (i.e., cabbage, cauliflower, brussels sprout). In this (the so-called ‘addiction hypothesis’).236 Thus, even a partial case, it was shown that several of antiapoptotic genes, whose inhibition of this pathway might be sufficient to negatively affect expression is controlled by NF-kB (XIAP, cIAP-1, cIAP-2, cFLIP, neoplastic survival and proliferation while sparing normal cells. TRAF-1), were suppressed by indole-3-carbinol.226 Obviously, it will be of the outmost importance the selection of As to synthetic inhibitors, SN50, a peptide which blocks patients with molecular dependence on the pathway. 227 nuclear import of NF-kB, decreased TRAIL or As2O3 resis- How could the biological effectiveness of PI3K/Akt pathway tance of HL60AR cells, as shown by our laboratory.118,119 A inhibitor be measured in vivo? At variance with patients with recent study has highlighted that AS602868, a small molecule solid tumors, the evaluation of the efficacy of the therapeutic which selectively targets IKKb, induced apoptosis of AML blasts treatment should be more easily feasible in AML patients, using and also potentiated the apoptotic response induced by either peripheral blood or bone marrow samples. Existing assays chemotherapeutic drugs currently used for the treatment of that could be used to determine pathway inhibition include AML, such as doxorubicin, cytarabine and etoposide.121 BAY- immunohistochemistry with phosphospecific or native antibo- 11–7082 is yet another synthetic NF-kB inhibitor which caused dies recognizing levels of active or total protein, respec- apoptosis in human leukemic cell lines.228 tively,54,233,237 kinase assays,57 and/or flow cytometry using All these studies have validated NF-kB as a promising the same antibodies whenever possible.56,57,238 Flow cytometry therapeutic target downstream of PI3K/Akt in AML. It may also appears particularly well suited for evaluation of AML patients, be worth mentioning here that bortezomib, an indirect NF-kB as it offers obvious advantages over the other techniques inhibitor which targets the proteasome,229 has been approved (especially Western blot and kinase assays), including quick- for treatment of multiple myeloma230 and is currently under ness, a lower number of cells required to perform the assay, and clinical evaluation for other hematological malignancies, the possibility of identifying different subclones in the leukemic including AML.231 Interestingly, bortezomib induced apoptosis populations by coimmunostaining with multiple antibodies to of bone marrow monuclear myeloid (BMMM) cells from patients surface antigens, even when the percentage of blast cells is with high-risk MDS, a preneoplastic condition which frequently usually low such as in some AML subgroups (FAB M2, M4, develops into overt AML, and is characterized by a progressive M5).57,239,240 The use of this technique is now greatly facilitated increase in bone marrow blasts with reduced apoptotic by the availability of fluorochrome-conjugated primary anti- capacities.232 In this connection, our laboratory has very bodies directed to phosphoproteins, including Akt, and by the recently shown that BMMM cells from high-risk MDS patients possibility of performing immunostaining in the whole blood.241 frequently display high levels of phosphorylated Akt, which Moreover, flow cytometry would be extremely well suited for might be responsible for NF-kB upregulation.233 the analysis of the effects of treatment on the rare LSCs, the real However, a cautionary note is required here. Although NF-kB target for AML eradication. activation in most cases is harmful, there is evidence that NF-kB The side effects reported so far in patients treated with upregulation has also beneficial effects. Indeed, NF-kBis inhibitors of this pathway (CCI-779, perifosine, UCN-01) required for normal functioning of the , for include thrombocytopenia, anemia, liver disfunction, hypocal- hematopoiesis, and could even promote apoptosis,234 for cemia, maculopapular rash, mucositis, hyperglycemia, nausea, example by positively regulating the expression of TRAIL DR5 vomiting and diarrhea.202,242,243 The occurrence of hypergly- receptor.235 Therefore, NF-kB inhibitors should be tested with cemia raises the issue of impaired glucose tolerance and type II caution in view of the dual nature of this transcription factor. diabetes, which might be caused by inhibition of this signaling network. These findings are in agreement with the data showing significant increase in insulin serum levels of animal treated Concluding remarks and future directions with indazole-pyridine Akt inhibitors.186 Even though blood glucose levels were normal, the increase in serum insulin is A growing excitement surrounds the development of signal consistent with a homeostatic response, where the animals transduction modulators as new powerful agents for treating secrete more insulin to maintain blood glucose concentrations

Leukemia PI3K/Akt signaling in AML AM Martelli et al 922 and oppose insulin resistance. This is also what has been However, the continuing efforts to develop specific, high-affinity observed in Akt2 knockout mice.33 When young, these animals PI3K/Akt inhibitors will surely yield new effective drugs. have normal blood glucose concentrations but increased plasma Intelligent clinical trial design, coupled with rigorous scientific insulin. Only when older, after pancreatic islet b-cell failure, do evaluation, may herald entering a new era in which signal the animals lose their ability to maintain normal blood glucose transduction modulators could significantly improve the out- levels. This indicates that PI3K/Akt inhibitors might induce type come of AML. II diabetes in patients who would be treated for a prolonged period of time or who are elder. In this connection, a key issue that has not been adequately addressed in in vitro studies, let Acknowledgements alone in in vivo studies, is how long the pathway will need to be inhibited to cause cell cycle arrest or apoptosis of cancer cells? This work was supported by grants from: Italian MIUR FIRB 2001 Obviously, shorter exposures in patients might be associated and COFIN 2005, Associazione Italiana Ricerca sul Cancro (AIRC with less toxicity. However, we do not know whether or not Regional Grants), CARISBO Foundation. continual exposure to inhibitory compounds could be tolerated, and whether similar responses could be achieved with long- term, low-dose exposure versus short-term, high-dose exposure. References As to the occurrence of thrombocytopenia and anemia, this might suggest detrimental effects of the inhibitors on hemato- 1 Smith M, Barnett M, Bassan R, Gatta G, Tondini C, Kern W. poiesis. This may appear surprising, because, according to the Adult acute myeloid leukaemia. Crit Rev Oncol Hematol 2004; literature, phosphorylated Akt is extremely low or absent in 50: 197–222. 54,233,237 2 Ferrara F. Unanswered questions in acute myeloid leukaemia. normal bone marrow. Furthermore, several investiga- Lancet Oncol 2004; 5: 443–450. tions have shown that PI3K/Akt inhibitors are much less toxic for 3 Tallman MS, Gilliland DG, Rowe JM. Drug therapy for acute normal human hematopoietic precursor cells than for AML myeloid leukemia. Blood 2005; 106: 1154–1163. blasts.23,71,121,204 However, it should not been overlooked that 4 Lowenberg B, Downing JR, Burnett A. Acute myeloid leukemia. many activate PI3K/Akt signaling which therefore N Engl J Med 1999; 341: 1051–1062. would seem to play an important role in erythropoiesis, 5 Grimwade D, Enver T. Acute promyelocytic leukemia: where 244–247 does it stem from? Leukemia 2004; 18: 375–384. myelopoiesis and thrombocytopoiesis. Clearly, these 6 Taussig DC, Pearce DJ, Simpson C, Rohatiner AZ, Lister TA, Kelly results have been obtained in vitro and they might not reflect G et al. Hematopoietic stem cells express multiple myeloid what happens in vivo. In addition, also in this field the findings markers: implications for the origin and targeted therapy of acute are conflicting, as exemplified by a recent paper which suggests myeloid leukemia. Blood 2005; 106: 4086–4092. that EPO downregulates PI3K/Akt axis during erythropoiesis 7 Warner JK, Wang JC, Takenaka K, Doulatov S, McKenzie JL, through upregulation of phosphatidylinositol 4-phosphatase Harrington L et al. Direct evidence for cooperating genetic events 248 in the leukemic transformation of normal human hematopoietic II, whereas other reports have suggested a key role for cells. Leukemia 2005; 19: 1794–1805. 245,249,250 PI3K/Akt in EPO-mediated erythroid differentiation. 8 Sambani C, La Starza R, Roumier C, Crescenzi B, Stavropoulou C, One of the challenges that lie ahead is understanding whether Katsarou O et al. Partial duplication of the MLL oncogene in PI3K/Akt inhibitors will be effective in vivo when employed patients with aggressive acute myeloid leukemia. Haematologica alone or will require to be combined with other treatments, such 2004; 89: 403–407. as chemotherapy. Conceivably, a combination therapy would 9 Tonks A, Tonks AJ, Pearn L, Pearce L, Hoy T, Couzens S et al. Expression of AML1-ETO in human myelomonocytic cells allow the use of lower dosage of signal transduction modulators selectively inhibits granulocytic differentiation and promotes giving maximum efficacy and minimum side effects, as their self-renewal. Leukemia 2004; 18: 1238–1245. substantial evidence is accumulating for synergy between kinase 10 Nanri T, Matsuno N, Kawakita T, Suzushima H, Kawano F, inhibition and classical chemotherapy. Mitsuya H et al. Mutations in the receptor tyrosine kinase Finally, what component would be the best target in such a pathway are associated with clinical outcome in patients with heavily branched signaling network? Would it be preferable to acute myeloblastic leukemia harboring t(8;21)(q22;q22). Leuke- mia 2005; 19: 1361–1366. target single components of the branches further downstream of 11 Shih LY, Huang CF, Wang PN, Wu JH, Lin TL, Dunn P et al. PI3K/Akt, such as mTOR or NF-kB, that are more exclusively Acquisition of FLT3 or N-ras mutations is frequently associated involved in cell growth, proliferation and survival? Or would with progression of myelodysplastic syndrome to acute myeloid ‘cocktails’ of drugs affecting multiple steps of the pathway to be leukemia. Leukemia 2004; 18: 466–475. an even more effective form of therapy? Indeed, the PI3K/Akt 12 Yanada M, Matsuo K, Suzuki T, Kiyoi H, Naoe T. Prognostic pathway bifurcates and integrates with other signaling networks significance of FLT3 internal tandem duplication and tyrosine kinase domain mutations for acute myeloid leukemia: a meta- as the signal is propagated. The pathway could therefore be analysis. Leukemia 2005; 19: 1345–1349. more globally, and thereby effectively, inhibited when also 13 Cammenga J. Gatekeeper pathways and cellular background targeting PI3K or Akt. At least in theory, this kind of inhibition in the pathogenesis and therapy of AML. Leukemia 2005; 19: might also be less susceptible to the complicating and still 1719–1728. largely ill-defined effects of feedback loops than the inhibition of 14 Moore MA. Converging pathways in leukemogenesis and stem single branches downstream. However, this efficacy would cell self-renewal. Exp Hematol 2005; 33: 719–737. 15 Brazil DP, Yang ZZ, Hemmings BA. Advances in presumably be balanced by potential for higher toxicity and a signalling: AKT ion on multiple fronts. Trends Biochem Sci 2004; narrower therapeutic index. 29: 233–242. The last several years have witnessed major progress towards 16 Hanada M, Feng J, Hemmings BA. Structure, regulation and the goal of translating our growing understanding of the function of PKB/AKT – a major therapeutic target. Biochim molecular basis of cancer into drugs with improved therapeutic Biophys Acta 2004; 1697: 3–16. activity and selectivity. Tremendous advances have been made 17 Bellacosa A, Kumar CC, Di Cristofano A, Testa JR. Activation of AKT kinases in cancer: implications for therapeutic targeting. but significant obstacles remain. Additional work addressing all Adv Cancer Res 2005; 94: 29–86. the aforementioned issues is needed to determine if the potential 18 Osaki M, Oshimura M, Ito H. PI3K-Akt pathway: its functions and of PI3K/Akt inhibitors may be fully realized in AML treatment. alterations in human cancer. Apoptosis 2004; 9: 667–676.

Leukemia PI3K/Akt signaling in AML AM Martelli et al 923 19 Song G, Ouyang G, Bao S. The activation of Akt/PKB signaling activated by daunorubicin in human acute myeloid leukemia cell pathway and cell survival. J Cell Mol Med 2005; 9: 59–71. lines. FEBS Lett 1999; 452: 150–154. 20 Kim D, Dan HC, Park S, Yang L, Liu Q, Kaneko S et al. AKT/PKB 44 Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: a link between signaling mechanisms in cancer and chemoresistance. Front cancer genetics and chemotherapy. Cell 2002; 108: 153–164. Biosci 2005; 10: 975–987. 45 Zauli G, Sancilio S, Cataldi A, Sabatini N, Bosco D, Di Pietro R. 21 Kubota Y, Ohnishi H, Kitanaka A, Ishida T, Tanaka T. Constitutive PI-3K/Akt and NF-kB/IkBa pathways are activated in Jurkat T cells activation of PI3K is involved in the spontaneous proliferation in response to TRAIL treatment. J Cell Physiol 2005; 202: 900– of primary acute myeloid leukemia cells: direct evidence of PI3K 911. activation. Leukemia 2004; 18: 1438–1440. 46 Steelman LS, Pohnert SC, Shelton JG, Franklin RA, Bertrand FE, 22 Min YH, Eom JI, Cheong JW, Maeng HO, Kim JY, Jeung HK et al. McCubrey JA. JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in Constitutive phosphorylation of Akt/PKB protein in acute myeloid cell cycle progression and leukemogenesis. Leukemia 2004; 18: leukemia: its significance as a prognostic variable. Leukemia 189–218. 2003; 17: 995–997. 47 Backers K, Blero D, Paternotte N, Zhang J, Erneux C. The 23 Xu Q, Simpson SE, Scialla TJ, Bagg A, Carroll M. Survival of acute termination of PI3K signalling by SHIP1 and SHIP2 inositol myeloid leukemia cells requires PI3 kinase activation. Blood 5-phosphatases. Adv Regul 2003; 43: 15–28. 2003; 102: 972–980. 48 Sansal I, Sellers WR. The biology and clinical relevance of the 24 Zhao S, Konopleva M, Cabreira-Hansen M, Xie Z, Hu W, Milella PTEN tumor suppressor pathway. J Clin Oncol 2004; 22: 2954– M et al. Inhibition of phosphatidylinositol 3-kinase depho- 2963. sphorylates BAD and promotes apoptosis in myeloid leukemias. 49 Choi Y, Zhang J, Murga C, Yu H, Koller E, Monia BP et al. PTEN, Leukemia 2004; 18: 267–275. but not SHIP and SHIP2, suppresses the PI3K/Akt pathway and 25 Stauffer F, Holzer P, Garcia-Echeverria C. Blocking the PI3K/PKB induces growth inhibition and apoptosis of myeloma cells. pathway in tumor cells. Curr Med Chem Anti-Canc Agents 2005; Oncogene 2002; 21: 5289–5300. 5: 449–462. 50 Janssens V, Goris J, Van Hoof C. PP2A: the expected tumor 26 Vanhaesebroeck B, Ali K, Bilancio A, Geering B, Foukas LC. suppressor. Curr Opin Genet Dev 2005; 15: 34–41. Signalling by PI3K isoforms: insights from gene-targeted mice. 51 Borgatti P, Martelli AM, Tabellini G, Bellacosa A, Capitani S, Neri Trends Biochem Sci 2005; 30: 194–204. LM. Threonine 308 phosphorylated form of Akt translocates to the 27 Anderson KE, Jackson SP. Class I phosphoinositide 3-kinases. Int J nucleus of PC12 cells under nerve growth factor stimulation and Biochem Cell Biol 2003; 35: 1028–1033. associates with the nuclear matrix protein nucleolin. J Cell 28 Stephens L, Williams R, Hawkins P. Phosphoinositide 3-kinases Physiol 2003; 196: 79–88. as drug targets in cancer. Curr Opin Pharmacol 2005; 5: 357– 52 Liu W, Akhand AA, Takeda K, Kawamoto Y, Itoigawa M, Kato M 365. et al. Protein phosphatase 2A-linked and -unlinked caspase- 29 Hennessy BT, Smith DL, Ram PT, Lu Y, Mills GB. Exploiting the dependent pathways for downregulation of Akt kinase triggered PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug by 4-hydroxynonenal. Cell Death Differ 2003; 10: 772–781. Discov 2005; 4: 988–1004. 53 Gao T, Furnari F, Newton AC. PHLPP: a phosphatase that directly 30 Wymann MP, Bjorklof K, Calvez R, Finan P, Thomast M, Trifilieff dephosphorylates Akt, promotes apoptosis, and suppresses tumor A et al. Phosphoinositide 3-kinase gamma: a key modulator growth. Mol Cell 2005; 18: 13–24. in inflammation and allergy. Biochem Soc Trans 2003; 31: 54 Brandts CH, Sargin B, Rode M, Biermann C, Lindtner B, Schwable 275–280. J et al. Constitutive activation of Akt by Flt3 internal tandem 31 Franke TF, Hornik CP, Segev L, Shostak GA, Sugimoto C. duplications is necessary for increased survival, proliferation, and PI3K/Akt and apoptosis: size matters. Oncogene 2003; 22: myeloid transformation. Cancer Res 2005; 65: 9643–9650. 8983–8998. 55 Grandage VL, Gale RE, Linch DC, Khwaja A. PI3-kinase/Akt is 32 Steelman LS, Bertrand FE, McCubrey JA. The complexity of PTEN: constitutively active in primary acute myeloid leukaemia cells mutation, marker and potential target for therapeutic intervention. and regulates survival and chemoresistance via NF-kappaB, Expert Opin Ther Targets 2004; 8: 537–550. Mapkinase and p53 pathways. Leukemia 2005; 19: 586–594. 33 Cho H, Mu J, Kim JK, Thorvaldsen JL, Chu Q, Crenshaw III EB 56 Kornblau SM, Womble M, Cade JS, Lemker E, Qiu YH. et al. Insulin resistance and a diabetes mellitus-like syndrome in Comparative analysis of the effects of sample source and test mice lacking the protein kinase Akt2 (PKB b). Science 2001; 292: methodology on the assessment of protein expression in acute 1728–1731. myelogenous leukemia. Leukemia 2005; 19: 1550–1557. 34 Chen WS, Xu PZ, Gottlob K, Chen ML, Sokol K, Shiyanova T et al. 57 Tazzari PL, Cappellini A, Grafone T, Mantovani I, Ricci F, Billi Growth retardation and increased apoptosis in mice with AM et al. Detection of serine 473 phosphorylated Akt in acute homozygous disruption of the Akt1 gene. Genes Dev 2001; 15: myeloid leukaemia blasts by flow cytometry. Br J Haematol 2004; 2203–2208. 126: 675–681. 35 Easton RM, Cho H, Roovers K, Shineman DW, Mizrahi M, 58 Birkenkamp KU, Geugien M, Schepers H, Westra J, Lemmink Forman MS et al. Role for Akt3/protein kinase Bg in attainment of HH, Vellenga E. Constitutive NF-kB DNA-binding activity in normal brain size. Mol Cell Biol 2005; 25: 1869–1878. AML is frequently mediated by a Ras/PI3-K/PKB-dependent 36 Sarbassov DD, Guertin DA, Ali SM, Sabatini DM. Phosphoryla- pathway. Leukemia 2004; 18: 103–112. tion and regulation of Akt/PKB by the rictor-mTOR complex. 59 Minami Y, Yamamoto K, Kiyoi H, Ueda R, Saito H, Naoe T. Science 2005; 307: 1098–1101. Different antiapoptotic pathways between wild-type and mutated 37 Di Maira G, Salvi M, Arrigoni G, Marin O, Sarno S, Brustolon F FLT3: insights into therapeutic targets in leukemia. Blood 2003; et al. Protein kinase CK2 phosphorylates and upregulates Akt/ 102: 2969–2975. PKB. Cell Death Differ 2005; 12: 668–677. 60 Scheijen B, Ngo HT, Kang H, Griffin JD. FLT3 receptors with 38 Ye K. PIKE/nuclear PI 3-kinase signaling in preventing pro- internal tandem duplications promote cell viability and prolifera- grammed cell death. J Cell Biochem 2005; 96: 463–472. tion by signaling through Foxo proteins. Oncogene 2004; 23: 39 Du K, Tsichlis PN. Regulation of the Akt kinase by interacting 3338–3349. proteins. Oncogene 2005; 24: 7401–7409. 61 Choudhary C, Schwable J, Brandts C, Tickenbrock L, Sargin B, 40 Pearl LH. Hsp90 and Cdc37 – a chaperone cancer conspiracy. Kindler T et al. AML-associated Flt3 kinase domain mutations Curr Opin Genet Dev 2005; 15: 55–61. show signal transduction differences compared with Flt3 ITD 41 West KA, Castillo SS, Dennis PA. Activation of the PI3K/Akt mutations. Blood 2005; 106: 265–273. pathway and chemotherapeutic resistance. Drug Resist Updat 62 Schwable J, Choudhary C, Thiede C, Tickenbrock L, Sargin B, 2002; 5: 234–248. Steur C et al. RGS2 is an important target gene of Flt3-ITD 42 Pelicano H, Carney D, Huang P. ROS stress in cancer cells and mutations in AML and functions in myeloid differentiation and therapeutic implications. Drug Resist Updat 2004; 7: 97–110. leukemic transformation. Blood 2005; 105: 2107–2114. 43 Plo I, Bettaieb A, Payrastre B, Mansat-De Mas V, Bordier C, 63 Noguera NI, Ammatuna E, Zangrilli D, Lavorgna S, Divona M, Rousse A et al. The phosphoinositide 3-kinase/Akt pathway is Buccisano F et al. Simultaneous detection of NPM1 and FLT3-ITD

Leukemia PI3K/Akt signaling in AML AM Martelli et al 924 mutations by capillary electrophoresis in acute myeloid leuke- of genes with relevant functions in the different subclasses of mia. Leukemia 2005; 19: 1479–1482. acute myeloid leukemia. Leukemia 2005; 19: 402–409. 64 Naoe T, Kiyoi H. Normal and oncogenic FLT3. Cell Mol Life Sci 86 List AF, Glinsmann-Gibson B, Stadheim C, Meuillet EJ, Bellamy 2004; 61: 2932–2938. W, Powis G. Vascular endothelial growth factor receptor-1 and 65 Frohling S, Scholl C, Gilliland DG, Levine RL. Genetics of receptor-2 initiate a phosphatidylinositide 3-kinase-dependent myeloid malignancies: pathogenetic and clinical implications. J clonogenic response in acute myeloid leukemia cells. Exp Clin Oncol 2005; 23: 6285–6295. Hematol 2004; 32: 526–535. 66 Roskoski Jr R. Structure and regulation of Kit protein-tyrosine 87 Wakabayashi M, Miwa H, Shikami M, Hiramatsu A, Ikai T, kinase – the stem cell factor receptor. Biochem Biophys Res Tajima E et al. Autocrine pathway of angiopoietins-Tie2 system in Commun 2005; 338: 1307–1315. AML cells: association with phosphatidyl-inositol 3 kinase. 67 Kohl TM, Schnittger S, Ellwart JW, Hiddemann W, Spiekermann Hematol J 2004; 5: 353–360. K. KIT exon 8 mutations associated with core-binding factor 88 Neri LM, Borgatti P, Tazzari PL, Bortul R, Cappellini A, Tabellini (CBF)-acute myeloid leukemia (AML) cause hyperactivation of the G et al. The phosphoinositide 3-kinase/AKT1 pathway involve- receptor in response to stem cell factor. Blood 2005; 105: 3319– ment in drug and all-trans-retinoic acid resistance of leukemia 3321. cells. 68 Ning ZQ, Li J, McGuinness M, Arceci RJ. STAT3 activation is Mol Cancer Res 2003; 1: 234–246. required for Asp(816) mutant c-Kit induced tumorigenicity. 89 Estrov Z, Meir R, Barak Y, Zaizov R, Zadik Z. Human growth Oncogene 2001; 20: 4528–4536. hormone and insulin-like growth factor-1 enhance the prolifera- 69 Larizza L, Magnani I, Beghini A. The Kasumi-1 cell line: a t(8;21)- tion of human leukemic blasts. J Clin Oncol 1991; 9: 394–399. kit mutant model for acute myeloid leukemia. Leuk Lymphoma 90 Grimberg A. Mechanisms by which IGF-I may promote cancer. 2005; 46: 247–255. Cancer Biol Ther 2003; 2: 630–635. 70 Beghini A, Bellini M, Magnani I, Colapietro P, Cairoli R, Morra E 91 Mitsiades CS, Mitsiades N, Poulaki V, Schlossman R, Akiyama M, et al. STI 571 inhibition effect on KITAsn822Lys-mediated signal Chauhan D et al. Activation of NF-kappaB and upregulation transduction cascade. Exp Hematol 2005; 33: 682–688. of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling 71 Sujobert P, Bardet V, Cornillet-Lefebvre P, Hayflick JS, Prie N, in human multiple myeloma cells: therapeutic implications. Verdier F et al. Essential role for the p110d isoform in Oncogene 2002; 21: 5673–5683. phosphoinositide 3-kinase activation and cell proliferation in 92 Matsunaga T, Takemoto N, Sato T, Takimoto R, Tanaka I, Fujimi acute myeloid leukemia. Blood 2005; 106: 1063–1066. A et al. Interaction between leukemic-cell VLA-4 and stromal 72 Sawyer C, Sturge J, Bennett DC, O’Hare MJ, Allen WE, Bain J fibronectin is a decisive factor for minimal residual disease of et al. Regulation of breast cancer cell chemotaxis by the acute myelogenous leukemia. Nat Med 2003; 9: 1158–1165. phosphoinositide 3-kinase p110d. Cancer Res 2003; 63: 1667– 93 De Toni F, Racaud-Sultan C, Chicanne G, Mas VM, Cariven C, 1675. Mesange F et al. A crosstalk between the Wnt and the adhesion- 73 Cornillet-Lefebvre P, Cuccuini W, Bardet V, Tamburini J, Gillot L, dependent signaling pathways governs the chemosensitivity of Ifrah N et al. Constitutive phosphoinositide 3-kinase activation in acute myeloid leukemia. Oncogene 2006, in press. acute myeloid leukemia is not due to p110d mutations. Leukemia 94 Xu Q, Thompson JE, Carroll M. mTOR regulates cell survival 2006; 20: 374–376. after etoposide treatment in primary AML cells. Blood 2005; 106: 74 Bousquet M, Recher C, Queleen C, Demur C, Payrastre B, 4261–4268. Brousset P. Assessment of somatic mutations in phosphatidylino- 95 Thompson JE, Thompson CB. Putting the rap on Akt. J Clin Oncol sitol 2004; 22: 4217–4226. 3-kinase gene in human lymphoma and acute leukaemia. Br J 96 Basu S, Totty NF, Irwin MS, Sudol M, Downward J. Akt Haematol 2005; 131: 411–413. phosphorylates the Yes-associated protein, YAP, to induce 75 Samuels Y, Velculescu VE. Oncogenic mutations of PIK3CA in interaction with 14-3-3 and attenuation of p73-mediated human cancers. Cell Cycle 2004; 3: 1221–1224. apoptosis. Mol Cell 2003; 11: 11–23. 76 Samuels Y, Ericson K. Oncogenic PI3K and its role in cancer. 97 Tamm I, Wagner M, Schmelz K. Decitabine activates specific Curr Opin Oncol 2006; 18: 77–82. caspases downstream of p73 in myeloid leukemia. Ann Hematol 77 Cheong JW, Eom JI, Maeng HY, Lee ST, Hahn JS, Ko YW et al. 2005; 84 (Suppl 13): 47–53. Phosphatase and tensin homologue phosphorylation in the 98 Rizzo MG, Giombini E, Diverio D, Vignetti M, Sacchi A, Testa U C-terminal regulatory domain is frequently observed in acute et al. Analysis of p73 expression pattern in acute myeloid myeloid leukaemia and associated with poor clinical outcome. Br leukemias: lack of DeltaN-p73 expression is a frequent feature of J Haematol 2003; 122: 454–456. acute promyelocytic leukemia. Leukemia 2004; 18: 1804–1809. 78 Vazquez F, Grossman SR, Takahashi Y, Rokas MV, Nakamura N, 99 Olesen LH, Aggerholm A, Andersen BL, Nyvold CG, Guldberg P, Sellers WR. Phosphorylation of the PTEN tail acts as an inhibitory Norgaard JM et al. Molecular typing of adult acute myeloid switch by preventing its recruitment into a protein complex. J Biol leukaemia: significance of translocations, tandem duplications, Chem 2001; 276: 48627–48630. methylation, and selective gene expression profiling. Br J 79 Liu TC, Lin PM, Chang JG, Lee JP, Chen TP, Lin SF. Mutation Haematol 2005; 131: 457–467. analysis of PTEN/MMAC1 in acute myeloid leukemia. Am J 100 van Stijn A, van der Pol MA, Kok A, Bontje PM, Roemen GM, Hematol 2000; 63: 170–175. Beelen RH et al. Differences between the CD34+and CD34-blast 80 Aggerholm A, Gronbaek K, Guldberg P, Hokland P. Mutational compartments in apoptosis resistance in acute myeloid leukemia. analysis of the tumour suppressor gene MMAC1/PTEN in Haematologica 2003; 88: 497–508. malignant myeloid disorders. Eur J Haematol 2000; 65: 109–113. 101 Brozovic A, Fritz G, Christmann M, Zisowsky J, Jaehde U, Osmak 81 Dahia PL, Aguiar RC, Alberta J, Kum JB, Caron S, Sill H et al. M et al. Long-term activation of SAPK/JNK, p38 kinase and PTEN is inversely correlated with the cell survival factor Akt/PKB fas-L expression by cisplatin is attenuated in human carcinoma and is inactivated via multiple mechanismsin haematological cells that acquired drug resistance. Int J Cancer 2004; 112: malignancies. Hum Mol Genet 1999; 8: 185–193. 974–985. 82 Luo JM, Yoshida H, Komura S, Ohishi N, Pan L, Shigeno K et al. 102 Dai Y, Rahmani M, Pei XY, Khanna P, Han SI, Mitchell C et al. Possible dominant-negative mutation of the SHIP gene in acute Farnesyltransferase inhibitors interact synergistically with the myeloid leukemia. Leukemia 2003; 17: 1–8. Chk1 inhibitor UCN-01 to induce apoptosis in human leukemia 83 Yamamoto M, Suzuki Y, Kihira H, Miwa H, Kita K, Nagao M et al. cells through interruption of both Akt and MEK/ERK pathways and Expressions of four major protein Ser/Thr phosphatases in human activation of SEK1/JNK. Blood 2005; 105: 1706–1716. primary leukemic cells. Leukemia 1999; 13: 595–600. 103 O’Gorman DM, McKenna SL, McGahon AJ, Cotter TG. Inhibition 84 Scavelli C, Vacca A, Di Pietro G, Dammacco F, Ribatti D. of PI3-kinase sensitises HL60 human leukaemia cells to both Crosstalk between angiogenesis and lymphangiogenesis in tumor chemotherapeutic drug- and Fas-induced apoptosis by a JNK progression. Leukemia 2004; 18: 1054–1058. independent pathway. Leuk Res 2001; 25: 801–811. 85 Gutierrez NC, Lopez-Perez R, Hernandez JM, Isidro I, Gonzalez 104 Lunghi P, Tabilio A, Pinelli S, Valmadre G, Ridolo E, Albertini R B, Delgado M et al. Gene expression profile reveals deregulation et al. Expression and activation of SHC/MAP kinase pathway

Leukemia PI3K/Akt signaling in AML AM Martelli et al 925 in primary acute myeloid leukemia blasts. Hematol J 2001; 2: 125 Diehl JA, Cheng M, Roussel MF, Sherr CJ. Glycogen synthase 70–80. kinase-3b regulates cyclin D1 proteolysis and subcellular 105 Zhou BP, Hung MC. Novel targets of Akt, p21(Cipl/WAF1), and localization. Genes Dev 1998; 12: 3499–3511. MDM2. Semin Oncol 2002; 29: 62–70. 126 Medema RH, Kops GJ, Bos JL, Burgering BM. AFX-like Forkhead 106 Mayo LD, Donner DB. The PTEN, Mdm2, p53 tumor suppressor- transcription factors mediate cell-cycle regulation by Ras and oncoprotein network. Trends Biochem Sci 2002; 27: 462–467. PKB through p27kip1. Nature 2000; 404: 782–787. 107 Wattel E, Preudhomme C, Hecquet B, Vanrumbeke M, Quesnel 127 Seoane J, Le HV, Shen L, Anderson SA, Massague J. Integration B, Dervite I et al. p53 mutations are associated with resistance to of Smad and forkhead pathways in the control of neuro- chemotherapy and short survival in hematologic malignancies. epithelial and glioblastoma cell proliferation. Cell 2004; 117: Blood 1994; 84: 3148–3157. 211–223. 108 Greer EL, Brunet A. FOXO transcription factors at the interface 128 Kops GJ, Medema RH, Glassford J, Essers MA, Dijkers PF, Coffer between longevity and tumor suppression. Oncogene 2005; 24: PJ et al. Control of cell cycle exit and entry by protein kinase 7410–7425. B-regulated forkhead transcription factors. Mol Cell Biol 2002; 109 Arden KC, Biggs III WH. Regulation of the FoxO family of 22: 2025–2036. transcription factors by phosphatidylinositol-3 kinase-activated 129 Ramaswamy S, Nakamura N, Sansal I, Bergeron L, Sellers WR. signaling. Arch Biochem Biophys 2002; 403: 292–298. A novel mechanism of gene regulation and tumor suppression 110 Van Der Heide LP, Hoekman MF, Smidt MP. The ins and outs of by the transcription factor FKHR. Cancer Cell 2002; 2: 81–91. FoxO shuttling: mechanisms of FoxO translocation and transcrip- 130 Schmidt M, Fernandez de Mattos S, van der Horst A, Klomp- tional regulation. Biochem J 2004; 380: 297–309. maker R, Kops GJ, Lam EW et al. Cell cycle inhibition by FoxO 111 Cappellini A, Tabellini G, Zweyer M, Bortul R, Tazzari PL, Billi forkhead transcription factors involves downregulation of cyclin AM et al. The phosphoinositide 3-kinase/Akt pathway regulates D. Mol Cell Biol 2002; 22: 7842–7852. cell cycle progression of HL60 human leukemia cells through 131 Wendel HG, De Stanchina E, Fridman JS, Malina A, Ray S, Kogan cytoplasmic relocalization of the cyclin-dependent kinase S et al. Survival signalling by Akt and eIF4E in oncogenesis and inhibitor p27(Kip1) and control of cyclin D1 expression. cancer therapy. Nature 2004; 428: 332–337. Leukemia 2003; 17: 2157–2167. 132 Fingar DC, Blenis J. Target of rapamycin (TOR): an integrator of 112 Cheong JW, Eom JI, Maeng HY, Lee ST, Hahn JS, Ko YW et al. nutrient and growth factor signals and coordinator of cell growth Constitutive phosphorylation of FKHR transcription factor as a and cell cycle progression. Oncogene 2004; 23: 3151–3171. prognostic variable in acute myeloid leukemia. Leuk Res 2003; 133 Tokunaga C, Yoshino K, Yonezawa K. mTOR integrates amino 27: 1159–1162. acid- and energy-sensing pathways. Biochem Biophys Res 113 Gupta P, Niehans GA, LeRoy SC, Gupta K, Morrison VA, Schultz Commun 2004; 313: 443–446. C et al. Fas ligand expression in the bone marrow in 134 Martin DE, Soulard A, Hall MN. TOR regulates ribosomal protein myelodysplastic syndromes correlates with FAB subtype and gene expression via PKA and the Forkhead transcription factor anemia, and predicts survival. Leukemia 1999; 13: 44–53. FHL1. Cell 2004; 119: 969–979. 114 Lee JJ, Chung IJ, Park MR, Ryang DW, Park CS, Kim HJ. Increased 135 Giles FJ, Albitar M. Mammalian target of rapamycin as a therapeutic target in leukemia. Curr Mol Med 2005; 5: 653–661. angiogenesis and Fas-ligand expression are independent 136 Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC. processes in acute myeloid leukemia. Leuk Res 2001; 25: Identification of the tuberous sclerosis complex-2 tumor suppres- 1067–1073. sor gene product tuberin as a target of the phosphoinositide 115 Aggarwal BB. Nuclear factor-kB: the enemy within. Cancer Cell 3-kinase/akt pathway. Mol Cell 2002; 10: 151–162. 2004; 6: 203–208. 137 Zhang Y, Gao X, Saucedo LJ, Ru B, Edgar BA, Pan D. Rheb is a 116 Shishodia S, Aggarwal BB. Nuclear factor-kappaB activation: a direct target of the tuberous sclerosis tumour suppressor proteins. question of life or death. J Biochem Mol Biol 2002; 35: 28–40. Nat Cell Biol 2003; 5: 578–581. 117 Hayden MS, Ghosh S. Signaling to NF-kB. Genes Dev 2004; 18: 138 Hay N. The Akt-mTOR tango and its relevance to cancer. Cancer 2195–2224. Cell 2005; 8: 179–183. 118 Bortul R, Tazzari PL, Cappellini A, Tabellini G, Billi AM, Bareggi 139 Granville CA, Memmott RM, Gills JJ, Dennis PA. Handicapping R et al. Constitutively active Akt1 protects HL60 leukemia cells the race to develop inhibitors of the phosphoinositide 3-kinase/ from TRAIL-induced apoptosis through a mechanism involving Akt/mammalian target of rapamycin pathway. Clin Cancer Res k NF- B activation and cFLIP(L) up-regulation. Leukemia 2003; 17: 2006; 12: 679–689. 379–389. 140 Hardt SE, Sadoshima J. Glycogen synthase kinase-3b: a novel 119 Tabellini G, Cappellini A, Tazzari PL, Fala F, Billi AM, Manzoli L regulator of cardiac hypertrophy and development. Circ Res et al. Phosphoinositide 3-kinase/Akt involvement in arsenic 2002; 90: 1055–1063. trioxide resistance of human leukemia cells. J Cell Physiol 141 Prunier C, Hocevar BA, Howe PH. Wnt signaling: physiology and 2005; 202: 623–634. pathology. Growth Factors 2004; 22: 141–150. 120 Baumgartner B, Weber M, Quirling M, Fischer C, Page S, Adam 142 Muller-Tidow C, Steffen B, Cauvet T, Tickenbrock L, Ji P, M et al. Increased IkB kinase activity is associated with acti- Diederichs S et al. Translocation products in acute myeloid vated NF-kappaB in acute myeloid blasts. Leukemia 2002; 16: leukemia activate the Wnt signaling pathway in hematopoietic 2062–2071. cells. Mol Cell Biol 2004; 24: 2890–2904. 121 Frelin C, Imbert V, Griessinger E, Peyron AC, Rochet N, Philip P 143 Simon M, Grandage VL, Linch DC, Khwaja A. Constitutive et al. Targeting NF-kB activation via pharmacologic inhibition activation of the Wnt/b-catenin signalling pathway in acute of IKK2-induced apoptosis of human acute myeloid leukemia myeloid leukaemia. Oncogene 2005; 24: 2410–2420. cells. Blood 2005; 105: 804–811. 144 Tickenbrock L, Schwable J, Wiedehage M, Steffen B, Sargin B, 122 Sherr CJ, Roberts JM. CDK inhibitors: positive and negative Choudhary C et al. Flt3 tandem duplication mutations cooperate regulators of G1-phase progression. Genes Dev 1999; 13: with Wnt signaling in leukemic signal transduction. Blood 2005; 1501–1512. 105: 3699–3706. 123 Min YH, Cheong JW, Kim JY, Eom JI, Lee ST, Hahn JS et al. 145 O’Gorman DM, McKenna SL, McGahon AJ, Knox KA, Cotter TG. Cytoplasmic mislocalization of p27Kip1 protein is associated Sensitisation of HL60 human leukaemic cells to cytotoxic drug- with constitutive phosphorylation of Akt or protein kinase B induced apoptosis by inhibition of PI3-kinase survival signals. and poor prognosis in acute myelogenous leukemia. Cancer Res Leukemia 2000; 14: 602–611. 2004; 64: 5225–5231. 146 Puccetti E, Ruthardt M. Acute promyelocytic leukemia: 124 Min YH, Cheong JW, Lee MH, Kim JY, Lee ST, Hahn JS et al. PML/RARa and the leukemic stem cell. Leukemia 2004; 18: Elevated S-phase kinase-associated protein 2 protein expression 1169–1175. in acute myelogenous leukemia: its association with constitutive 147 Ramos AM, Fernandez C, Amran D, Sancho P, de Blas E, Aller P. phosphorylation of phosphatase and tensin homologue protein Pharmacologic inhibitors of PI3K/Akt potentiate the apoptotic and poor prognosis. Clin Cancer Res 2004; 10: 5123–5130. action of the antileukemic drug arsenic trioxide via glutathione

Leukemia PI3K/Akt signaling in AML AM Martelli et al 926 depletion and increased peroxide accumulation in myeloid inhibitor gefitinib in A-549 human non-small cell lung cancer leukemia cells. Blood 2005; 105: 4013–4020. xenografts. Mol Cancer Ther 2005; 4: 1349–1357. 148 Tabellini G, Tazzari PL, Bortul R, Evangelisti C, Billi AM, Grafone 169 Sadhu C, Masinovsky B, Dick K, Sowell CG, Staunton DE. T et al. Phosphoinositide 3-kinase/Akt inhibition increases arsenic Essential role of phosphoinositide 3-kinase delta in neutrophil trioxide-induced apoptosis of acute promyelocytic and T-cell directional movement. J Immunol 2003; 170: 2647–2654. leukaemias. Br J Haematol 2005; 130: 716–725. 170 Mora A, Komander D, van Aalten DM, Alessi DR. PDK1, the 149 Sanz MA, Fenaux P, Lo Coco F. Arsenic trioxide in the treatment master regulator of AGC kinase signal transduction. Semin Cell of acute promyelocytic leukemia. A review of current evidence. Dev Biol 2004; 15: 161–170. Haematologica 2005; 90: 1231–1235. 171 Daub H, Specht K, Ullrich A. Strategies to overcome resistance to 150 Bouralexis S, Findlay DM, Evdokiou A. Death to the bad guys: targeted protein kinase inhibitors. Nat Rev Drug Discov 2004; 3: targeting cancer via Apo2L/TRAIL. Apoptosis 2005; 10: 35–51. 1001–1010. 151 Kaufmann SH, Steensma DP. On the TRAIL of a new therapy for 172 Sato S, Fujita N, Tsuruo T. Interference with PDK1-Akt survival leukemia. Leukemia 2005; 19: 2195–2202. signaling pathway by UCN-01 (7-hydroxystaurosporine). Onco- 152 Cappellini A, Mantovani I, Tazzari PL, Grafone T, Martinelli G, gene 2002; 21: 1727–1738. Cocco L et al. Application of flow cytometry to molecular 173 Komander D, Kular GS, Bain J, Elliott M, Alessi DR, Van Aalten medicine: detection of tumor necrosis factor-related apoptosis- DM. Structural basis for UCN-01 (7-hydroxystaurosporine) inducing ligand receptors in acute myeloid leukaemia blasts. Int J specificity and PDK1 (3-phosphoinositide-dependent protein Mol Med 2005; 16: 1041–1048. kinase-1) inhibition. Biochem J 2003; 375: 255–262. 153 Riccioni R, Pasquini L, Mariani G, Saulle E, Rossini A, Diverio D 174 Sampath D, Cortes J, Estrov Z, Du M, Shi Z, Andreeff M et al. et al. TRAIL decoy receptors mediate resistance of acute myeloid Pharmacodynamics of cytarabine alone and in combination with leukemia cells to TRAIL. Haematologica 2005; 90: 612–624. 7-hydroxystaurosporine (UCN-01) in AML blasts in vitro and 154 Guo F, Sigua C, Tao J, Bali P, George P, Li Y et al. Cotreat- during a clinical trial. Blood 2006; 107: 2517–2524. ment with histone deacetylase inhibitor LAQ824 enhances 175 Senderowicz AM. Small-molecule cyclin-dependent kinase Apo-2L/tumor necrosis factor-related apoptosis inducing ligand- modulators. Oncogene 2003; 22: 6609–6620. induced death inducing signaling complex activity and apop- 176 Gills JJ, Dennis PA. The development of phosphatidylinositol tosis of human acute leukemia cells. Cancer Res 2004; 64: ether lipid analogues as inhibitors of the serine/threonine kinase, 2580–2589. Akt. Expert Opin Investig Drugs 2004; 13: 787–797. 155 Kataoka T. The caspase-8 modulator c-FLIP. Crit Rev Immunol 177 Kumar CC, Madison V. AKT crystal structure and AKT-specific 2005; 25: 31–58. inhibitors. Oncogene 2005; 24: 7493–7501. 156 Tamm I, Kornblau SM, Segall H, Krajewski S, Welsh K, Kitada S 178 Castillo SS, Brognard J, Petukhov PA, Zhang C, Tsurutani J, et al. Expression and prognostic significance of IAP-family genes Granville CA et al. Preferential inhibition of Akt and killing of in human cancers and myeloid leukemias. Clin Cancer Res 2000; Akt-dependent cancer cells by rationally designed phosphatidy- linositol ether lipid analogues. Cancer Res 2004; 64: 2782–2792. 6: 1796–1803. 179 Meuillet EJ, Ihle N, Baker AF, Gard JM, Stamper C, Williams R 157 Carter BZ, Kornblau SM, Tsao T, Wang RY, Schober WD, Milella et al. In vivo molecular pharmacology and antitumor activity of M et al. Caspase-independent cell death in AML: caspase the targeted Akt inhibitor PX-316. Oncol Res 2004; 14: 513–527. inhibition in vitro with pan-caspase inhibitors or in vivo by XIAP 180 Martelli AM, Tazzari PL, Tabellini G, Bortul R, Billi AM, Manzoli or Survivin does not affect cell survival or prognosis. Blood 2003; L et al. A new selective AKT pharmacological inhibitor reduces 102: 4179–4186. resistance to chemotherapeutic drugs, TRAIL, all-trans-retinoic 158 Carter BZ, Milella M, Tsao T, McQueen T, Schober WD, Hu W acid, and ionizing radiation of human leukemia cells. Leukemia et al. Regulation and targeting of antiapoptotic XIAP in acute 2003; 17: 1794–1805. myeloid leukemia. Leukemia 2003; 17: 2081–2089. 181 Tabellini G, Tazzari PL, Bortul R, Billi AM, Conte R, Manzoli L 159 Cai D, Wang Y, Ottmann OG, Barth PJ, Neubauer A, Burchert A. et al. Novel 2’-substituted, 3’-deoxy-phosphatidyl-myo-inositol FLT3-ITD-, but not BCR/ABL-transformed cells require concurrent analogues reduce drug resistance in human leukaemia cell lines Akt/mTor blockage to undergo apoptosis after histone deacety- with an activated phosphoinositide 3-kinase/Akt pathway. Br J lase inhibitor treatment. Blood 2006; 107: 2094–2097. Haematol 2004; 126: 574–582. 160 Schmidt-Arras D, Schwable J, Bohmer FD, Serve H. Flt3 receptor 182 Kondapaka SB, Singh SS, Dasmahapatra GP, Sausville EA, Roy tyrosine kinase as a drug target in leukemia. Curr Pharm Des KK. Perifosine, a novel alkylphospholipid, inhibits protein kinase 2004; 10: 1867–1883. B activation. Mol Cancer Ther 2003; 2: 1093–1103. 161 Levis M. Recent advances in the development of small-molecule 183 Chun KH, Kosmeder II JW, Sun S, Pezzuto JM, Lotan R, Hong WK inhibitors for the treatment of acute myeloid leukemia. Curr Opin et al. Effects of deguelin on the phosphatidylinositol 3-kinase/Akt Hematol 2005; 12: 55–61. pathway and apoptosis in premalignant human bronchial 162 Morgan MA, Ganser A, Reuter CW. Therapeutic efficacy of epithelial cells. J Natl Cancer Inst 2003; 95: 291–302. prenylation inhibitors in the treatment of myeloid leukemia. 184 Barnett SF, Defeo-Jones D, Fu S, Hancock PJ, Haskell KM, Jones Leukemia 2003; 17: 1482–1498. RE et al. Identification and characterization of pleckstrin- 163 Moehler TM, Hillengass J, Goldschmidt H, Ho AD. Antiangio- homology-domain-dependent and isoenzyme-specific Akt inhi- genic therapy in hematologic malignancies. Curr Pharm Des bitors. Biochem J 2005; 385: 399–408. 2004; 10: 1221–1234. 185 DeFeo-Jones D, Barnett SF, Fu S, Hancock PJ, Haskell KM, 164 Garcia-Echeverria C, Pearson MA, Marti A, Meyer T, Mestan J, Leander KR et al. Tumor cell sensitization to apoptotic stimuli Zimmermann J et al. In vivo antitumor activity of NVP-AEW541 – by selective inhibition of specific Akt/PKB family members. a novel, potent, and selective inhibitor of the IGF-IR kinase. Mol Cancer Ther 2005; 4: 271–279. Cancer Cell 2004; 5: 231–239. 186 Luo Y, Shoemaker AR, Liu X, Woods KW, Thomas SA, de Jong R 165 Luo J, Manning BD, Cantley LC. Targeting the PI3K-Akt pathway et al. Potent and selective inhibitors of Akt kinases slow the in human cancer: rationale and promise. Cancer Cell 2003; 4: progress of tumors in vivo. Mol Cancer Ther 2005; 4: 977–986. 257–262. 187 Yang L, Dan HC, Sun M, Liu Q, Sun XM, Feldman RI et al. Akt/ 166 Martelli AM, Tabellini G, Bortul R, Tazzari PL, Cappellini A, Billi protein kinase B signaling inhibitor-2, a selective small molecule AM et al. Involvement of the phosphoinositide 3-kinase/Akt inhibitor of Akt signaling with antitumor activity in cancer cells signaling pathway in the resistance to therapeutic treatments of overexpressing Akt. Cancer Res 2004; 64: 4394–4399. human leukemias. Histol Histopathol 2005; 20: 239–252. 188 Shin I, Edl J, Biswas S, Lin PC, Mernaugh R, Arteaga CL. 167 Cheng JQ, Lindsley CW, Cheng GZ, Yang H, Nicosia SV. The Proapoptotic activity of cell-permeable anti-Akt single-chain Akt/PKB pathway: molecular target for cancer drug discovery. antibodies. Cancer Res 2005; 65: 2815–2824. Oncogene 2005; 24: 7482–7492. 189 Bortul R, Tazzari PL, Billi AM, Tabellini G, Mantovani I, 168 Ihle NT, Paine-Murrieta G, Berggren MI, Baker A, Tate WR, Wipf Cappellini A et al. Deguelin, A PI3K/AKT inhibitor, enhances P et al. The phosphatidylinositol-3-kinase inhibitor PX-866 chemosensitivity of leukaemia cells with an active PI3K/AKT overcomes resistance to the epidermal growth factor receptor pathway. Br J Haematol 2005; 129: 677–686.

Leukemia PI3K/Akt signaling in AML AM Martelli et al 927 190 Rahmani M, Reese E, Dai Y, Bauer C, Payne SG, Dent P et al. ATP by inhibition of glycolysis in lymphoma and leukemia cells. Coadministration of histone deacetylase inhibitors and perifosine Leukemia 2005; 19: 2153–2158. synergistically induces apoptosis in human leukemia cells 210 Elstrom RL, Bauer DE, Buzzai M, Karnauskas R, Harris MH, through Akt and ERK1/2 inactivation and the generation of Plas DR et al. Akt stimulates aerobic glycolysis in cancer cells. ceramide and reactive oxygen species. Cancer Res 2005; 65: Cancer Res 2004; 64: 3892–3899. 2422–2432. 211 Belozerov VE, Van Meir EG. Hypoxia inducible factor-1: a novel 191 Hideshima T, Catley L, Yasui H, Ishitsuka K, Raje N, Mitsiades C target for cancer therapy. Anticancer Drugs 2005; 16: 901–909. et al. Perifosine, an oral bioactive novel alkylphospholipid, 212 Jiang BH, Jiang G, Zheng JZ, Lu Z, Hunter T, Vogt PK. inhibits Akt and induces in vitro and in vivo cytotoxicity in Phosphatidylinositol 3-kinase signaling controls levels of hypo- human multiple myeloma cells. Blood 2006, in press. xia-inducible factor 1. Cell Growth Differ 2001; 12: 363–369. 192 Crul M, Rosing H, de Klerk GJ, Dubbelman R, Traiser M, Reichert 213 Fisher TL, White MF. Signaling pathways: the benefits of good S et al. Phase I and pharmacological study of daily oral communication. Curr Biol 2004; 14: R1005–R1007. administration of perifosine (D-21266) in patients with advanced 214 Harrington LS, Findlay GM, Lamb RF. Restraining PI3K: mTOR solid tumours. Eur J Cancer 2002; 38: 1615–1621. signalling goes back to the membrane. Trends Biochem Sci 2005; 193 Yang ZZ, Tschopp O, Baudry A, Dummler B, Hynx D, Hemmings 30: 35–42. BA. Physiological functions of protein kinase B/Akt. Biochem Soc 215 Shi Y, Yan H, Frost P, Gera J, Lichtenstein A. Mammalian target of Trans 2004; 32: 350–354. rapamycin inhibitors activate the AKT kinase in multiple 194 Neckers L, Ivy SP. Heat shock protein 90. Curr Opin Oncol 2003; myeloma cells by up-regulating the insulin-like growth factor 15: 419–424. receptor/insulin receptor substrate-1/phosphatidylinositol 3-ki- 195 Kamal A, Thao L, Sensintaffar J, Zhang L, Boehm MF, Fritz LC nase cascade. Mol Cancer Ther 2005; 4: 1533–1540. et al. A high-affinity conformation of Hsp90 confers tumour 216 Mitsiades CS, Mitsiades NS, McMullan CJ, Poulaki V, Shringar- selectivity on Hsp90 inhibitors. Nature 2003; 425: 407–410. pure R, Akiyama M et al. Inhibition of the insulin-like growth 196 Minami Y, Kiyoi H, Yamamoto Y, Yamamoto K, Ueda R, Saito H factor receptor-1 tyrosine kinase activity as a therapeutic strategy et al. Selective apoptosis of tandemly duplicated FLT3-trans- for multiple myeloma, other hematologic malignancies, and solid formed leukemia cells by Hsp90 inhibitors. Leukemia 2002; 16: tumors. Cancer Cell 2004; 5: 221–230. 1535–1540. 217 Miller DS, Fricker G, Drewe J. p-Glycoprotein-mediated transport 197 George P, Bali P, Cohen P, Tao J, Guo F, Sigua C et al. of a fluorescent rapamycin derivative in renal proximal tubule. Cotreatment with 17-allylamino-demethoxygeldanamycin and J Pharmacol Exp Ther 1997; 282: 440–444. FLT-3 kinase inhibitor PKC412 is highly effective against human 218 van der Kolk DM, de Vries EG, Noordhoek L, van den Berg E, van acute myelogenous leukemia cells with mutant FLT-3. Cancer der Pol MA, Muller M et al. Activity and expression of the multidrug resistance proteins P-glycoprotein, MRP1, MRP2, Res 2004; 64: 3645–3652. 198 George P, Bali P, Annavarapu S, Scuto A, Fiskus W, Guo F et al. MRP3 and MRP5 in de novo and relapsed acute myeloid leukemia. Leukemia 2001; 15: 1544–1553. Combination of the histone deacetylase inhibitor LBH589 and the 219 Guzman ML, Neering SJ, Upchurch D, Grimes B, Howard DS, hsp90 inhibitor 17-AAG is highly active against human CML-BC Rizzieri DA et al. Nuclear factor-kB is constitutively activated in cells and AML cells with activating mutation of FLT-3. Blood primitive human acute myelogenous leukemia cells. Blood 2001; 2005; 105: 1768–1776. 98: 2301–2307. 199 Yu W, Rao Q, Wang M, Tian Z, Lin D, Liu X et al. The Hsp90 220 Guzman ML, Swiderski CF, Howard DS, Grimes BA, Rossi RM, inhibitor 17-allylamide-17-demethoxygeldanamycin induces Szilvassy SJ et al. Preferential induction of apoptosis for primary apoptosis and differentiation of Kasumi-1 harboring the human leukemic stem cells. Proc Natl Acad Sci USA 2002; 99: Asn822Lys KIT mutation and down-regulates KIT protein level. 16220–16225. Leuk Res 2006; 30: 575–582. 221 Dorai T, Aggarwal BB. Role of chemopreventive agents in cancer 200 Mesa RA, Loegering D, Powell HL, Flatten K, Arlander SJ, Dai NT therapy. Cancer Lett 2004; 215: 129–140. et al. Heat shock protein 90 inhibition sensitizes acute 222 Kuo ML, Huang TS, Lin JK. Curcumin, an antioxidant and anti- myelogenous leukemia cells to cytarabine. Blood 2005; 106: tumor promoter, induces apoptosis in human leukemia cells. 318–327. Biochim Biophys Acta 1996; 1317: 95–100. 201 Hresko RC, Mueckler M. mTOR.RICTOR is the Ser473 kinase for 223 Anto RJ, Mukhopadhyay A, Denning K, Aggarwal BB. Curcumin Akt/protein kinase B in 3T3-L1 adipocytes. J Biol Chem 2005; (diferuloylmethane) induces apoptosis through activation of 280: 40406–40416. caspase-8, BID cleavage and cytochrome c release: its suppres- 202 Dutcher JP. Mammalian target of rapamycin inhibition. Clin sion by ectopic expression of Bcl-2 and Bcl-xl. Carcinogenesis Cancer Res 2004; 10: 6382S–6387S. 2002; 23: 143–150. 203 Huang S, Houghton PJ. Targeting mTOR signaling for cancer 224 Ito K, Nakazato T, Yamato K, Miyakawa Y, Yamada T, Hozumi N therapy. Curr Opin Pharmacol 2003; 3: 371–377. et al. Induction of apoptosis in leukemic cells by homovanillic 204 Recher C, Beyne-Rauzy O, Demur C, Chicanne G, Dos Santos C, acid derivative, capsaicin, through oxidative stress: implication of Mas VM et al. Antileukemic activity of rapamycin in acute phosphorylation of p53 at Ser-15 residue by reactive oxygen myeloid leukemia. Blood 2005; 105: 2527–2534. species. Cancer Res 2004; 64: 1071–1078. 205 Hahn M, Li W, Yu C, Rahmani M, Dent P, Grant S. Rapamycin 225 Estrov Z, Shishodia S, Faderl S, Harris D, Van Q, Kantarjian HM and UCN-01 synergistically induce apoptosis in human leukemia et al. Resveratrol blocks interleukin-1b-induced activation of the cells through a process that is regulated by the Raf-1/MEK/ERK, nuclear transcription factor NF-kB, inhibits proliferation, causes Akt, and JNK signal transduction pathways. Mol Cancer Ther S-phase arrest, and induces apoptosis of acute myeloid leukemia 2005; 4: 457–470. cells. Blood 2003; 102: 987–995. 206 Kaufmann SH, Karp JE, Svingen PA, Krajewski S, Burke PJ, Gore 226 Takada Y, Andreeff M, Aggarwal BB. Indole-3-carbinol sup- SD et al. Elevated expression of the apoptotic regulator Mcl-1 at presses NF-kB and IkappaBalpha kinase activation, causing the time of leukemic relapse. Blood 1998; 91: 991–1000. inhibition of expression of NF-kB-regulated antiapoptotic and 207 van Stijn A, Kok A, van der Pol MA, Feller N, Roemen GM, metastatic gene products and enhancement of apoptosis in Westra AH et al. A flow cytometric method to detect apoptosis- myeloid and leukemia cells. Blood 2005; 106: 641–649. related protein expression in minimal residual disease in acute 227 Lin YZ, Yao SY, Veach RA, Torgerson TR, Hawiger J. Inhibition myeloid leukemia. Leukemia 2003; 17: 780–786. of nuclear translocation of transcription factor NF-k Bbya 208 Yu C, Rahmani M, Dai Y, Conrad D, Krystal G, Dent P et al. The synthetic peptide containing a cell membrane-permeable motif lethal effects of pharmacological cyclin-dependent kinase in- and nuclear localization sequence. J Biol Chem 1995; 270: hibitors in human leukemia cells proceed through a phosphati- 14255–14258. dylinositol 3-kinase/Akt-dependent process. Cancer Res 2003; 228 Hansson A, Marin YE, Suh J, Rabson AB, Chen S, Huberman E 63: 1822–1833. et al. Enhancement of TPA-induced growth inhibition and 209 Xu RH, Pelicano H, Zhang H, Giles FJ, Keating MJ, Huang P. apoptosis in myeloid leukemia cells by BAY 11–7082, an Synergistic effect of targeting mTOR by rapamycin and depleting NF-kappaB inhibitor. Int J Oncol 2005; 27: 941–948.

Leukemia PI3K/Akt signaling in AML AM Martelli et al 928 229 Horton TM, Gannavarapu A, Blaney SM, D’Argenio DZ, Plon SE, 240 Irish JM, Hovland R, Krutzik PO, Perez OD, Bruserud O, Gjertsen Berg SL. Bortezomib interactions with chemotherapy agents in BT et al. Single cell profiling of potentiated phospho-protein acute leukemia in vitro. Cancer Chemother Pharmacol 2006, in networks in cancer cells. Cell 2004; 118: 217–228. press. 241 Krutzik PO, Hale MB, Nolan GP. Characterization of the murine 230 Richardson PG, Sonneveld P, Schuster MW, Irwin D, Stadtmauer immunological signaling network with phosphospecific flow EA, Facon T et al. Bortezomib or high-dose dexamethasone cytometry. J Immunol 2005; 175: 2366–2373. for relapsed multiple myeloma. N Engl J Med 2005; 352: 242 Van Ummersen L, Binger K, Volkman J, Marnocha R, Tutsch K, 2487–2498. Kolesar J et al. A phase I trial of perifosine (NSC 639966) on a 231 Orlowski RZ, Voorhees PM, Garcia RA, Hall MD, Kudrik FJ, loading dose/maintenance dose schedule in patients with Allred T et al. Phase 1 trial of the proteasome inhibitor advanced cancer. Clin Cancer Res 2004; 10: 7450–7456. bortezomib and pegylated liposomal doxorubicin in patients 243 Sausville EA, Arbuck SG, Messmann R, Headlee D, Bauer KS, with advanced hematologic malignancies. Blood 2005; 105: Lush RM et al. Phase I trial of 72-hour continuous infusion 3058–3065. UCN-01 in patients with refractory neoplasms. J Clin Oncol 232 Braun T, Carvalho G, Coquelle A, Vozenin MC, Lepelley P, 2001; 19: 2319–2333. Hirsch F et al. NF-kB constitutes a potential therapeutic target 244 Geddis AE, Fox NE, Kaushansky K. Phosphatidylinositol 3-kinase in high-risk myelodysplastic syndrome. Blood 2006; 107: is necessary but not sufficient for thrombopoietin-induced 1156–1165. proliferation in engineered Mpl-bearing cell lines as well as in 233 Nyakern M, Tazzari PL, Finelli C, Bosi C, Follo MY, Grafone T primary megakaryocytic progenitors. J Biol Chem 2001; 276: 34473–34479. et al. Frequent elevation of Akt kinase phosphorylation in 245 Myklebust JH, Blomhoff HK, Rusten LS, Stokke T, Smeland EB. blood marrow and peripheral blood mononuclear cells from Activation of phosphatidylinositol 3-kinase is important for high-risk myelodysplastic syndrome patients. Leukemia 2006; 20: erythropoietin-induced erythropoiesis from CD34(+) hemato- 230–238. poietic progenitor cells. Exp Hematol 2002; 30: 990–1000. k 234 Shishodia S, Aggarwal BB. Nuclear factor- B: a friend or a foe 246 Lewis JL, Marley SB, Ojo M, Gordon MY. Opposing effects of PI3 in cancer? Biochem Pharmacol 2004; 68: 1071–1080. kinase pathway activation on human myeloid and erythroid 235 Shetty S, Graham BA, Brown JG, Hu X, Vegh-Yarema N, Harding progenitor cell proliferation and differentiation in vitro. Exp G et al. Transcription factor NF-kB differentially regulates death Hematol 2004; 32: 36–44. receptor 5 expression involving histone deacetylase 1. Mol Cell 247 Young SM, Cambareri AC, Ashman LK. Role of c-KIT expression Biol 2005; 25: 5404–5416. level and phosphatidylinositol 3-kinase activation in survival and 236 Workman P. The opportunities and challenges of personalized proliferative responses of early myeloid cells. Cell Signal 2006; genome-based molecular therapies for cancer: targets, technol- 18: 608–620. ogies, and molecular chaperones. Cancer Chemother Pharmacol 248 Barnache S, Le Scolan E, Kosmider O, Denis N, Moreau-Gachelin 2003; 52 (Suppl 1): S45–S56. F. Phosphatidylinositol 4-phosphatase type II is an erythropoietin- 237 Hsu J, Shi Y, Krajewski S, Renner S, Fisher M, Reed JC et al. responsive gene. Oncogene 2006; 25: 1420–1423. The AKT kinase is activated in multiple myeloma tumor cells. 249 Ghaffari S, Kitidis C, Zhao W, Marinkovic D, Fleming MD, Luo B Blood 2001; 98: 2853–2855. et al. AKT induces erythroid-cell maturation of JAK2-deficient 238 Tazzari PL, Cappellini A, Bortul R, Ricci F, Billi AM, Tabellini G fetal liver progenitor cells and is required for Epo regulation of et al. Flow cytometric detection of total and serine 473 erythroid-cell differentiation. Blood 2006; 107: 1888–1891. phosphorylated Akt. J Cell Biochem 2002; 86: 704–715. 250 Zhao W, Kitidis C, Fleming MD, Lodish HF, Ghaffari S. 239 Krutzik PO, Nolan GP. Intracellular phospho-protein staining Erythropoietin stimulates phosphorylation and activation of techniques for flow cytometry: monitoring single GATA-1 via the PI3-kinase/AKT signaling pathway. Blood 2006; events. Cytometry A 2003; 55: 61–70. 107: 907–915.

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