Leading Article

Leukemia (2006) 20, 911–928. doi:10.1038/sj.leu.2404245; published online 27 April 2006

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

A M Martelli1,2, M Nyåkern1, G Tabellini3, R Bortul4, P L Tazzari5, C Evangelisti1 and L Cocco1

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

Correspondence: Dr AM Martelli, Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell'Apparato Locomotore, Sezione di Anatomia Umana, Cell Signalling Laboratory, Università di Bologna, Bologna 40126, Italy. E-mail: amartell@biocfarm.unibo.it

Received 23 February 2006; Accepted 27 March 2006; Published online 27 April 2006.

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Abstract

The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is crucial to many aspects of cell growth, survival and apoptosis, and its constitutive activation has been implicated in the both the pathogenesis and the progression of a wide variety of neoplasias. Hence, this pathway is an attractive target for the development of novel anticancer strategies. Recent studies showed that PI3K/Akt signaling is frequently activated in acute myeloid leukemia (AML) patient blasts and strongly contributes to proliferation, survival and drug resistance 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 molecules designed to selectively target key components of this signal transduction cascade induce apoptosis and/or markedly increase conventional drug sensitivity of AML blasts in vitro. Thus, inhibitory molecules are currently being developed for clinical use either as single agents or in combination with conventional therapies. However, the PI3K/Akt pathway is important for many physiological cellular functions and, in particular, for insulin signaling, so that its blockade in vivo might cause severe systemic side effects. In this review, we summarize the existing knowledge about PI3K/Akt signaling in AML cells and we examine the rationale for targeting this fundamental signal transduction network by means of selective pharmacological inhibitors.

Keywords:

signal transduction networks, 3-phosphorylated inositol lipids, apoptosis, drug resistance, targeted molecular therapy

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Introduction

Acute myeloid leukemia (AML) is a heterogeneous group of malignant hematopoietic disorders characterized by uncontrolled proliferation of clonal neoplastic cells and accumulation in the bone marrow of blasts with an impaired differentiation program which are blocked at various maturation steps and resistant to cell death. Acute myeloid leukemia accounts for approximately 80% of all adult leukemias and its overall incidence has been stable or slowly increasing over the last 15–20 years.1 Despite considerable advances in the diagnosis of the different AML subtypes and progress in therapeutic approaches, current chemotherapies produce only initial remission, so that most patients will relapse and die from the disease. The 5-year survival rate of AML has hovered at 15–30% since the 1970s,1, 2, 3 and patients with AML arising out of myelodysplastic syndrome (MDS) or who are older than 60 years do even worse.4 Therefore, there remains a need for new, rationally designed, minimally toxic, effective therapies for AML.

Several independent laboratories have demonstrated that AML arises from leukemic stem cells (LSCs) that have an extended phenotype lineage negative (lin-), CD34+, CD38-, CD123+, CD33+, CD13+/-.5, 6 Indeed, injection of these cells into immunocompromised nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice leads to a disease with phenotypic and molecular characteristics of human AML. Over the last few years, remarkable progress has been made in the elucidation of the molecular pathogenesis of AML. A recent 'two hits' model has suggested that AML development requires multiple genetic changes that deregulate different cell programs.7 Transcription factor fusion proteins such as AML1/ETO, PML-RARalpha, CBFbeta/MYH11 or MLL/AF9 block myeloid cell differentiation by repressing target genes, thus providing one necessary event for leukemogenesis.8, 9 Disordered cell growth and upregulation of cell survival genes is a proposed necessary second event. Mutations in growth regulatory genes such as FLT3, Ras and c-Kit are common in AML patients.10, 11, 12 The most recent data provide evidence of great interdependence between 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 receptors, whereas activating mutations in signal transduction molecules induce alterations in the activity and expression of several transcription factors that are essential for normal myeloid differentiation.13, 14

The phosphoinositide 3-kinase (PI3K)/Akt signaling network is crucial to widely divergent physiological processes that include cell cycle progression, differentiation, transcription, translation and apoptosis.15, 16 It is targeted by genomic aberrations including amplification, mutation and rearrangement more frequently than any other pathway in human cancer, with the possible exception of the p53 and retinoblastoma pathways. Activation of PI3K/Akt signaling results in disturbance of control of cell proliferation and apoptosis, ensuing in competitive growth advantage for tumor cells.17, 18, 19 Furthermore, it is now clear that PI3K/Akt axis upregulation may be one of the major factors undermining successful antineoplastic treatment, thus portending a poor prognosis in many cancer types.20 Therefore, the PI3K/Akt pathway is an attractive target for the development of novel therapeutic strategies in patients with various tumor types.

Several recent papers have highlighted that the PI3K/Akt axis is activated in AML.21, 22, 23, 24 Remarkably, both the disease-free survival and overall survival were significantly shorter in AML cases with upregulated PI3K/Akt pathway.25 Therefore, this signal transduction cascade may represent a valid target for innovative therapeutic treatments of AML patients. The main aim of this review is to discuss potential antineoplastic strategies targeting this signaling network in AML. However, we shall begin with a general outline of the mechanisms that govern PI3K/Akt activation and of the responses generated along this signaling cascade with a particular emphasis placed on AML.

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PI3K family of isozymes and their activation

The large family of PI3K lipid kinases in mammalian cells has been categorized into three classes, referred to as I, II and III, each of which has its own characteristics in terms of molecular structure and substrate specificity.26 Class I PI3Ks are the best understood and are key players of multiple intracellular signaling networks that integrate a wide variety of signals, engaged by many polypeptide growth factors. For this reason, they will be the only isoforms considered relevant to this review. Growth factor receptors drive activation of class I PI3Ks either directly or via associated tyrosine kinases, heterotrimeric G proteins or Ras. Class I PI3K preferred in vivo substrate is phosphatidylinositol 4,5 bisphosphate (PtdIns (4,5)P2), which is phosphorylated to yield phosphatidylinositol 3,4,5 trisphosphate (PtdIns (3,4,5)P3). They are further divided into class IA and IB PI3Ks. Class IA PI3Ks are composed of heterodimers of an adaptor/regulatory (either p85 or p55) and a p110 catalytic subunit.27 There are at least seven adaptor/regulatory proteins that are generated by expression and alternative splicing of three different genes (referred to as Pik3r1, Pik3r2 and Pik3r3), whereas three p110 isoforms have been identified: alpha, beta and delta.28 These are encoded by three different genes, PI3KCA, PI3KCB and PI3KCD. The adaptor/regulatory subunits act to localize PI3K to the plasma membrane by the interaction of their Src homology 2 (SH2) domains with phosphotyrosine residues in activated receptors. They also serve to stabilize p110 and to limit its activity. Insulin and some growth factors preferentially signal through p110beta.29

The single class IB PI3K or PI3Kitalic gamma is made of a p110italic gamma catalytic subunit and a p101 regulatory subunit, unrelated to p85. p110italic gamma signals downstream of heterotrimeric G proteins and Ras26 and its upregulation is a hallmark of inflammation.30

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Akt isoforms and their activation

Akt, a serine/threonine protein kinase also known as protein kinase B (PKB), is the mammalian homolog of the transforming viral oncogene v-Akt that causes murine T-cell lymphoma.31 Akt, which belongs to the AGC kinase superfamily, is now known to include three closely related, highly conserved isoforms encoded by the following distinct genetic loci: Akt1/alpha, Akt2/beta and Akt3/italic gamma.15, 16 Akt1 is ubiquitously expressed at high levels with the exception of the kidney, liver and spleen. Akt2 expression varies between different organs, with higher expression 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 relatively highly expressed in the brain and testis.31, 32 Gene knockout studies have defined the biological importance of Akt isoforms in normal cells. In particular, Akt2-null mice develop a typical type II diabetes,33 whereas Akt1- and Akt3-deficient mice are not diabetic but display a decrease in size of all the organs and a selective impairment of brain development, respectively.34, 35

Akt contains an NH2-terminal pleckstrin homology (PH) domain, which interacts with the phosphorylated lipid products of PI3K (mainly PtdIns (3,4,5)P3 and, to a lesser extent, phosphatidylinositol 3,4 bisphosphate (PtdIns (3,4,)P2)) synthesized at the plasma membrane. Akt recruitment at the plasma membrane results in a conformational change, which enables the activation loop of the kinase to be phosphorylated on Thr 308 by phosphoinositide-dependent protein kinase-1 (PDK-1, which also requires 3-phosphorylated inositol lipids for activation and plasma membrane translocation) and at Ser 473 in the C-terminal hydrophobic motif by a kinase (often referred to as PDK-2) whose identity, despite intense investigation, remains highly controversial.15, 16 Candidate PDK-2s include integrin-linked kinase, DNA-dependent protein kinase and mitogen-activated protein kinase-kinase 2. The corresponding phosphorylation sites of Akt2 are Thr 309 and Ser 474, whereas those of Akt3 are Thr 305 and Ser 472.15 Recent evidence has highlighted that Ser 473 phosphorylation precedes Thr 308 phosphorylation and actually enhances it (see Sarbassov et al.36 and references therein).

Furthermore, it has been shown that additional phosphorylative steps may increase Akt activation, including phosphorylation of multiple tyrosine residues,15 and phosphorylation on Ser 129 through casein kinase 2 (CK2),37 but relevance of these phosphorylative events awaits full elucidation. Phosphorylated Akt migrates to both the cytosol and the nucleus. Nuclear Akt may fullfil an important antiapoptotic role.38 However, the relative contribution of Akt signaling at the plasma membrane, the cytosol and the nucleus also remain to be determined.

For the scopes of this review, it may be worth mentioning here that Akt activity is modulated by a complex network of regulatory proteins that interact with the PH domain, or the kinase domain or the C-terminal of Akt.39 One of these proteins is heat-shock protein-90 (HSP-90), a molecular chaperone that forms a complex with the co-chaperone Cdc37. This complex binds a variety of proteins, including tyrosine and serine/threonine protein kinases.40 The HSP-90/Cdc37 complex interacts with the Akt kinase domain. Therefore, small molecules capable of disrupting such an interaction may represent valid drugs to block Akt function.

It should also be emphasized that PI3K/Akt signaling can be upregulated by many forms of cellular stress including heat shock, low pH, ultraviolet light, ischemia, hypoxia, hypoglycemia and oxidative stress.41 Stress-induced PI3K/Akt upregulation is to be viewed as a compensatory protective mechanism which cells activate for escaping death. This is very relevant to the topic of this review, because neoplastic cells perceive chemotherapy as an insult, and many types of chemotherapy exert their cytotoxic effects through the generation of reactive oxygen species.42 Consistently, it has been reported that daunorubicin rapidly upregulated the PI3K/Akt pathway in U937 human leukemia cells.43 The exact molecular mechanism underlying this activation is unclear, even though it is now well established that apoptogenic stimuli quite often initiate an antagonistic antiapoptotic program.44, 45

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Negative regulation of the PI3K/Akt pathway

As 3-phosphorylated inositides are not hydrolyzed by any known phospholipase C, a counter-regulation by phosphatases has emerged as a crucial process to control PI3K-dependent signaling. PTEN (Phosphatase and TENsin homolog deleted on chromosome 10) is a dual specificity lipid and protein phosphatase that preferentially removes the 3-phosphate mainly from PtdIns (3,4,5)P3 but is also active on PtdIns (3,4,)P2, thereby antagonizing PI3K/Akt signaling network.46 PTEN-inactivating mutations or silencing occur in a wide variety of human cancers (including glioblastoma, melanoma, prostatic and endometrium carcinomas) and this results in Akt upregulation.47 Therefore, PTEN is a tumor suppressor acting upstream of Akt.48 Two other phosphatases, SHIP-1 and SHIP-2 (for SH domain-containing inositol phosphatases), are capable of removing the 5-phosphate from PtdIns (3,4,5)P3 to yield PtdIns (3,4,)P2.49 Whereas SHIP-1 is predominantly expressed in hematopoietic cells, SHIP-2 is more ubiquitous. However, their role on Akt function is not well understood, and in some cases they could not reverse Akt activation, something PTEN can do.49 Protein phosphatase 2A (PP2A), which is rapidly emerging as a new oncosuppressor,50 is capable of directly dephosphorylating and downregulating Akt,51, 52 whereas recent work indicates that Ser 473 phospho-Akt is dephosphorylated by a PP2C family phosphatase, referred to as PHLPP, another candidate tumor suppressor.53

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Activation of the PI3K/Akt signaling network in AML

Recently, several papers have highlighted that constitutive activation of PI3K/Akt signaling is a common feature of AML.21, 22, 23, 24, 54, 55, 56, 57 From 50 to 70% of patients with AML display phosphorylation of both Thr 308 and Ser 473 Akt. No correlation was shown to exist between Akt phosphorylation levels and French–American–British (FAB) subtype of AML, percentage blast infiltration of the bone marrow, cytogenetic anomalies, or when comparing untreated versus relapsed/refractory AML.54 However, the overall survival time for patients demonstrating Akt activation was significantly shorter when compared to patients with no Akt activation.22 Although the mechanisms that upregulate PI3K/Akt signaling in AML cells remain unclear, Akt activation in AML blasts may be dependent on, or independent from, PI3K.21

In about 15–25% of AML cases, N-Ras or K-Ras gene point mutations have been detected. These mutations abrogate Ras intrinsic GTPase activity and lead to constitutive Ras activation with a consequent stimulatory effect on the PI3K/Akt pathway.58 Indeed, it is well established that Ras can activate the PI3K/Akt axis either by itself or through the Raf/MEK/ERK pathway.46

Up to 20–25% of AML patients harbor internal tandem duplication (ITD) of the juxtamembraen domain of FLT3. This mutation results in ligand-independent dimerization of FLT3 and constitutive upregulation of its tyrosine kinase activity, ensuing in stimulation of downstream signaling pathways, including PI3K/Akt.59 Surprisingly, however, most of the papers focusing on FLT3-ITD and PI3K/Akt upregulation show data obtained with mouse cells such as 32D or BaF3.60, 61, 62 Importance of FLT3-ITD in causing PI3K/Akt upregulation of mouse myeloid precursors is demonstrated by a study in which overexpression of FLT3-ITD cDNA resulted in constitutive activation of Akt, which phosphorylated and inhibited the transcription factor FoxO3 (see later). Restored FoxO3 activity reversed FLT3-ITD-mediated growth properties and dominant-negative Akt prevented FLT3-ITD-mediated cytokine independence of 32D myeloid precursors.54 Overall, there is need for investigations showing that in AML blasts FLT3-ITD directly correlates with PI3K/Akt upregulation.

It is worth remembering here that FLT3, a member of the class III receptor tyrosine kinases, is preferentially localized on the cell surface of hematopoietic progenitors, and its ligand (FL) is expressed as a membrane-bound or soluble form by bone marrow stroma cells. It has been disclosed that FL–FLT3 interaction plays an important role in the proliferation and differentiation of hematopoietic cells. FLT3 is also expressed in a high proportion of AML cells. Activating mutations of FLT3 are the second most frequent genetic lesions in AML (nucleophosmin mutations are now considered to be the most common, see Noguera et al.63), and AML patients with FLT3 mutations have a worse prognosis than those with normal FLT3.64 In addition, mutations of the FLT3 tyrosine kinase domain (FLT3-TKD) activation loop have been detected in a minority (7%) of patients with AML. However, their functional consequences are not well understood and so far they have not been shown to be of significant prognostic relevance.65 Moreover, in a mouse model of myeloid precursors, FLT3-TKD activation loop mutations were not associated with increased PI3K/Akt activity.61

About 80% of AML patients have blast cells that express c-Kit, another class III receptor tyrosine kinase for the stem cell factor (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 known for activating PI3K/Akt; however, these results have not been obtained with AML cell lines and/or blasts.67, 68 A gain-of-function point mutation of c-Kit (Asn822Lys) has been reported in the Kasumi-1 AML cell line, which carries the t(8;21) translocation. PI3K-dependent activation of Akt was observed in Kasumi-1 cells.69, 70 Therefore, the outcome of c-Kit mutations on PI3K/Akt signaling needs to be further explored in AML cell lines and blasts.

Moreover, it has been recently demonstrated that the PI3K p110delta catalytic subunit isoform was consistently expressed at high levels in AML blasts, in contrast to the other class I isoforms (alpha, beta), of which the expression was very variable among patients, even if, in some of them, was quite high. Interestingly, IC87114, a p110delta-selective pharmacological inhibitor, suppressed both constitutive and FL-stimulated Akt activation in AML blasts to the same extent as LY294002, a non-selective inhibitor of PI3K isozymes.71 However, the reason for higher expression of p110delta in AML blasts remains unclear. Originally, this isoform had been thought to be specific for cells of hematopoietic lineage, but more recently it has also been detected in some non-hematopoietic cell types, especially those of breast or melanocytic origin, both in the untransformed and transformed state.72

Conceivably, the above findings are not mutually exclusive, because activated Ras, FLT3 or c-Kit could impinge on elevated levels of p110delta, also considering that upregulation of PI3K p110delta activity in AML blasts does not seem to be dependent on activating mutations.73 No mutations have been found in the gene coding for p110alpha in AML blasts,74 whereas PI3KCA is frequently amplified or harbors activating mutations in several solid tumors, including gastric, colon, breast and liver cancer.75, 76

As to PTEN, a recent study highlighted that PTEN phosphorylation was present in approximately 75% of AML patients. PTEN phosphorylation was significantly associated with Akt phosphorylation and with shorter overall survival.77 It is known that phosphorylation at the C-terminal regulatory domain of PTEN stabilizes the molecule, but makes it less active towards its substrate, PtdIns (3,4,5)P3.78 Moreover, PTEN expression has been shown to be low or absent in some AML patients,23 although the level of PTEN expression did not always correlate with the degree of Akt phosphorylation. However, a subsequent study failed to demonstrate that AML blasts have a decreased expression of PTEN.55

A study of 62 AML patients, showed that 15 of them had aberrant PTEN transcripts. However, all the samples with abnormal transcripts also displayed normal full-length transcripts, suggesting that aberrant transcripts could result from altered RNA splicing. Morover, no loss of heterozygosity or mutations were found.79

As far as PTEN-inactivating mutations are concerned, they do not seem to occur very frequently in AML.80, 81 Therefore, the role, if any, of PTEN in causing Akt activation in AML blast cells is unclear.

As to other lipid or protein phosphatases, a study has implicated a dominant-negative SHIP-1 mutation as a possible cause of Akt activation in AML.82 Low levels of PP2A have also been reported in some AML types, but correlation with Akt activation is lacking.83

Other possible activation mechanisms of the PI3K/Akt cascade in AML cells have been recently proposed. Vascular endothelial growth factor (VEGF) is a powerful angiogenic molecule for hematological malignancies. It behaves as a critical regulator of endothelial cell survival, motility and proliferation.84 It is intriguing that AML blasts synthesize and secrete VEGF and have demonstrable VEGF receptors, that is, VEGFR-1 and VEGFR-2.85 Using KG1 and HL60 human leukemic cell lines as experimental models, it has been shown that VEGF elicited a rapid and sustained Akt phosphorylation through a mechanism which was dependent on PI3K because it could be inhibited by wortmannin.86 In addition, in AML blasts and human acute leukemia cell lines, angiopoietins activated PI3K through an autocrine mechanism.87 Therefore, at least in some AML cases, upregulation of the PI3K/Akt axis might be due to an autocrine and/or paracrine production of angiogenic factors, such as VEGF and angiopoietins.

Our laboratory has shown that in a HL60 cell line subclone, activation of PI3K/Akt signaling was dependent on autocrine secretion of insulin-like growth factor-1 (IGF-1).88 At present, there is no evidence that a similar mechanism could be effective in AML blasts, even though it is known that the growth of these cells is increased in vitro in response to IGF-1 stimulation.89 Nevertheless, we feel that this possibility should be further explored, because over the past 5 years multiple large case–control studies have reported positive associations between high circulating levels of IGF-1 and risk for different types of cancer.90 Regarding malignant hematopoietic disorders, the role of IGF-1 in promoting proliferation, survival and drug resistance of multiple myeloma cells through PI3K/Akt signaling is well established.91

Finally, interactions between very late antigen (VLA)-4 (alpha4beta1 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 aspects of this have been contested by another group.93

In Table 1 and Figure 1, we summarize the possible mechanisms of activation of PI3K/Akt signaling in AML cells.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

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 p110delta. 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.

Full figure and legend (172K)


Whatever the reason might be for PI3K/Akt activation, it is very important to emphasize that recent results have highlighted that upregulation of PI3K/Ak axis is present not only in the bulk of the AML cell population, but also in LSCs tranplanted in NOD/SCID mice,94 where it exerts a powerful prosurvival effect. This finding indicates that therapeutical targeting of the PI3K/Akt pathway has the potential for eradicating AML.

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Antiapoptotic targets of PI3K/Akt pathway

Because Akt is the prototypic kinase which promotes cellular survival to apoptotic insults, survival by Akt is the process that has been most intensely investigated. It is now clear that Akt enhances survival by directly phosphorylating key regulatory proteins of the apoptotic cascades. Akt phosphorylates Bad, a proapoptotic member of the Bcl-2 family, at Ser 136. This phosphorylation event promotes Bad sequestration by 14-3-3 proteins in the cytosol, thereby preventing Bad from interacting with either Bcl-2 or Bcl-XL at the mitochondrial membrane. The final effect is inhibition of apoptosis.95 Treatment with the PI3K inhibitor LY294002 reduced Ser 136 Bad phosphorylation and induced apoptosis of AML blasts with constitutively active PI3K/Akt pathway.24 This finding is an indication of the key role played by phosphorylated Bad to prevent apoptosis of AML cells. PI3K/Akt-dependent Bad phosphorylation has also been detected in HL60 leukemia cells.88 A similar negative regulation has been demonstrated for Yes-associated protein, whose phosphorylation by Akt leads to repression of p53-related transcription factor p73 and reduced expression of the proapoptotic protein Bax.96 Conflicting results exist in the literature as to p73 expression in AML blasts, because in some studies it has been reported that the p73 gene was not expressed owing to promoter hypermethylation,97 whereas in others p73 protein was detected in most of the investigated cases.98 Also, Bax expression in AML is a controversial issue, with some investigations reporting high levels and others demonstrating low levels.99, 100 Therefore, it is difficult at present to draw a firm conclusion about the relationship (if any) between PI3K/Akt activation and p73/Bax expression in AML blasts.

Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is an important mediator of apoptosis in cells exposed to a variety of noxious stimuli, including chemotherapeutic drugs.101 Akt may interfere with SAPK/JNK signaling and inhibit apoptosis by phosphorylating and thereby inactivating ASK1, a protein kinase which transduces signals to SAPK/JNK.16 In U937 leukemia cells, Akt signaling negatively regulated SAPK/JNK,102 at variance with previous results obtained with HL60 cells.103 Available evidence suggests that SAPK/JNK is not activated in AML blasts under basal conditions, but we could not exclude, it does in response to noxious stimuli such as chemotherapeutic drugs.104

In another mechanism to thwart apoptosis, Akt promotes phosphorylation and nuclear translocation of Mdm2, an E3 ubiquitin ligase which mediates ubiquitinylation and proteasome-dependent degradation of the p53 tumor suppressor protein,105, 106 thereby downregulating p53 and antagonizing p53-mediated cell cycle checkpoint. Consistently, blockade of the PI3K/Akt axis led to a more than twofold increase in p53 activity in four out of nine AML patients tested.55 This indicates that in some AML cases, p53 is regulated through PI3K/Akt-dependent signaling and this pathway could be a mechanism to promote resistance to cytotoxic agents.107

Moreover, Akt may promote cell survival by phosphorylating transcription factors that control the expression of pro- and anti-apoptotic genes. Akt either negatively affects factors that promote death gene expression or positively regulates factors inducing survival genes. An example of the former is the FoxO family of transcription factors, previously referred to as forkhead transcription factors.108 Phosphorylation of FoxO factors by Akt alters their intracellular localization: in the absence of Akt activation, FoxO proteins are predominantly localized in the nucleus where they are able to promote transcription of proapoptotic target genes such as Fas ligand (Fas-L) and Bim. Activation of the PI3K/Akt pathway leads to nuclear export of these transcription factors. In the nucleus, phospho-FoxO factors specifically interact with 14-3-3 proteins, which serve as chaperone molecules to escort them out of the nucleus. Once in the cytoplasm, they are degraded via the ubiquitin-proteasome pathway.109, 110 FoxO factor phosphorylation requires intranuclear localization of active Akt, which has been documented in both HL60 cells111 and AML blasts.54 Akt phosphorylation was found to be significantly associated with elevated levels of phospho-FoxO1 and phospho-FoxO3 (previously referred to as FKHR and FKHRL1, respectively) factors in 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. Although there is no information about the levels of Bim in AML patients, Fas-L has been found to be consistently upregulated in AML blasts.113, 114 This may indicate that in AML cells expression of this proapoptotic protein is not regulated through a PI3K/Akt/FoxO-dependent pathway.

In addition to downregulating FoxO activity, Akt is capable of upregulating nuclear factor-kappa B (NF-kappaB), a transcription factor which is deeply involved in the regulation of cell proliferation, apoptosis and survival.115, 116 The survival-promoting activity of NF-kappaB is mediated by its ability to induce expression of antiapoptotic proteins including cIAP-1 and -2, XIAP, c-FLIP and TRAFs, which oppose caspase activation. Members of the NF-kappaB family form dimers (classically heterodimers of p65 with p50) that, under non-stimulated conditions, are retained in the cytoplasm. NF-kappaB function is regulated through its association with the inhibitory cofactor I-kappaB, which sequesters NF-kappaB. Phosphorylation of I-kappaB by upstream kinases, referred to as IKKs, promotes its degradation via the ubiquitin-proteasome pathway. This, in turn, allows NF-kappaB nuclear translocation and upregulation of target genes.117 Akt phosphorylates directly and activates IKKalpha and, more importantly, it is believed to be essential for IKK-mediated destruction of I-kappaB.16 Our laboratory has shown that the PI3K/Akt axis regulates NF-kappaB-dependent gene expression in HL60 cells.118, 119 An increased level of activated nuclear NF-kappaB has also been reported in myeloid blasts120, 121 which is frequently mediated by the PI3K/Akt signaling network.55, 58

All in all, the importance of the PI3K/Akt pathway in determining survival of AML blasts is emphasized by the observation that a pharmacological inhibitor of this network (LY294002), when employed alone, greatly enhanced the apoptosis rate of AML cells in culture.23, 24

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PI3K/Akt and cell cycle regulation

Akt targets p27Kip1, a direct inhibitor of cyclin-dependent kinase (cdk) 2, one of the cdks responsible for the activation of E2F1 transcription factors that promote DNA replication.122 When phosphorylated by Akt on Thr 157, p27Kip1 mainly 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 3beta (GSK3beta) (see later), because cyclin D1 phosphorylation by GSK3beta results in its destabilization.125 However, enhanced cell proliferation could also be a consequence 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, 127, 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 p27Kip and p21Waf/Cip1 expression and activation of PI3K/Akt signaling in AML blasts.55 Therefore, how activation of the PI3K/Akt axis could positively influence proliferation of AML cells remains to be fully elucitated.

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PI3K/Akt and metabolism

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase which regulates translation in response to nutrients/growth factors by phosphorylating components of the protein synthesis machinery, including p70S6 kinase (p70S6K, a ribosomal kinase) and eukaryotic initiation factor (eIF)-4E binding protein (4EBP)-1, the latter resulting in release of the translation initiation factor eIF-4E, allowing eIF-4E to participate in assembly of a translational initiation complex.131 Conceivably, mTOR acts as a checkpoint sensor indicating to cells that there are sufficient nutrients available to proceed through the cell cycle.132, 133 Therefore, mTOR regulates a variety of steps involved in protein synthesis, but in particular favors the production of key molecules such as c-Myc, cyclin D1 and ribosomal proteins.134 p70S6K, which can also be directly activated by PDK-1, phosphorylates the 40s ribosomal protein, S6, leading to active translation of mRNAs.135 By controlling protein synthesis, p70S6K and 4E-BP1 also regulate cell growth and hypertrophy, which are important processes for neoplastic progression. Therefore, even more distal steps in the PI3K/Akt pathway may have the potential to be exploited for cancer treatment. Akt-mediated regulation of mTOR activity is a complex multistep phenomenon (Figure 2). Akt inhibits tuberous sclerosis 2 (TSC2 or hamartin) function through direct phosphorylation.136 Tuberous sclerosis 2 is a GTPase-activating protein (GAP) that functions in association with the putative TSC1 (or tuberin) to inactivate the small G protein Rheb (Ras homolog enriched in brain).137 Tuberous sclerosis 2 phosphorylation by Akt represses GAP activity of the TSC1/TSC2 complex, allowing Rheb to accumulate in a GTP-bound state. Rheb-GTP then activates through a mechanism not yet elucidated the protein kinase activity of mTOR when complexed with the Raptor (regulatory-associated protein of mTOR) adaptor protein and mLST8.138 The mTOR/Raptor/mLST8 complex is sensitive to rapamycin and, importantly, inhibits Akt via a negative feedback loop which involves, at least in part, p70S6K.138 The relationship between Akt and mTOR is further complicated by the existence of the mTOR/Rictor (rapamycin-insensitive companion of mTOR)/mLST8 complex, which displays rapamycin-insensitive activity. It should also be reminded here that Akt directly phosphorylates and activates mTOR, and this is to date the only example of Akt-mediated phosphorylation which results in substrate activation.139 mTOR was found to be phosphorylated in AML blasts, along with its two downstream substrates, p70S6K and 4EBP-1, in a PI3K/Akt-dependent fashion.23 Nevertheless, others failed to detect any relationship between PI3K/Akt upregulation and p70S6K phosphorylation in AML primary cells.55

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

The Akt/mTOR pathway. Active Akt inhibits TSC2 activity through direct phosphorylation. Tuberous sclerosis 2 is a GTPase-activating protein that functions in association with the putative TSC1 to inactivate the small G protein Rheb. Akt-driven TSC1/TSC2 complex inactivation allows Rheb to accumulate in a GTP-bound 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 downstream targets include p70S6K and 4E-BP1. Once phosphorylated by mTOR, 4E-BP1 dissociates from eIF-4E which can then initiate translation. However, mTOR also exists complexed with Rictor/mLST8. This complex phosphorylates Akt on Ser 473 and could be phosphoinositide-dependent protein kinase-2. Rapamycin binds the immunophilin FK506-binding protein 12 and then inactivates the mTOR/Raptor/mLSTB complex, but not the m/TOR/Rictor/mLST8 complex. Arrows indicate activating events, whereas perpendicular lines indicate inhibitory events.

Full figure and legend (20K)

Another Akt substrate important for metabolic function is GSK3beta, which phosphorylates and inactivates glycogen synthase in response to insulin stimulation. When phosphorylated by Akt on Ser 9, GSK3beta is downregulated.140 Glycogen synthase kinase 3beta is phosphorylated in AML cells with upregulated Akt function.77 However, as for p70S6K, others found that downregulation of PI3K/Akt signaling in AML primary cells did not result in GSK3beta dephosphorylation.55 Thus, further investigations are necessary to elucidate the role played by PI3K/Akt in controlling p70S6K and GSK3beta function in AML blasts.

Interestingly, GSK3beta has been implicated in the signaling pathway elicited by Wnt, a ligand for transmembrane receptor frizzled. The beta-catenin protein is at the core of the canonical Wnt signaling pathway. Wnt stimulation leads to beta-catenin accumulation, nuclear translocation and interaction with transcription factors to regulate genes important for embryonic development and proliferation.141 The Wnt/beta-catenin signaling network is constitutively activated in AML blasts as a result of the expression of transcription factor fusion proteins such as AML1/ETO142, 143 and there is evidence for neoplastic myeloid transformation supported by this pathway.144 Phosphorylation of beta-catenin by GSK3beta on key N-terminal residues targets it for ubiquitination and breakdown in the proteasome.143 Given that Akt phosphorylates and inactivates GSK3beta, upregulation of PI3K/Akt signaling in AML cells might increase the levels of beta-catenin resulting in its accumulation and translocation to the nucleus where it would stimulate the transcription of target genes that include c-Myc and cyclin D1. This may well be an example of interdependence between alterations in signaling pathways and changes in transcriptional activity of AML blasts, as outlined above.

Downstream targets of PI3K/Akt pathway identified in AML cell lines or blasts are summarized in Table 2.


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PI3K/Akt activation and AML resistance to therapeutic treatments

The largely acknowledged, fundamental role played by PI3K/Akt cascade in opposing apoptosis has led to an intense investigation into contribution of this signaling pathway to tumor cell survival in response to various types of therapeutic treatment. In a wide variety of neoplastic cells, PI3K/Akt signaling is deeply involved in resistance to classical antineoplastic chemotherapeutic agents including etoposide, anthracyclins and cisplatin.41 The importance of the PI3K/Akt network in causing resistance to drugs commonly used for AML treatment was first demonstrated by O'Gorman et al.145 These authors employed drug-resistant HL60 human leukemia cells to show that either wortmannin or LY294002 downregulated Akt activity and increased sensitivity to etoposide or doxorubicin. More recently, our group has confirmed these findings by taking advantage of a HL60 cell clone, referred to as HL60AR (apoptosis resistant cells) which, when compared with parental (PT) HL60 cells, displayed a constitutively active PI3K/Akt axis.88 HL60AR cells are much more resistant than HL60PT cells to a wide variety of chemotherapeutic drugs as well as to all-trans retinoic acid (ATRA), a powerful differentiating agent for HL60 cells that is successfully employed to treat acute promyelocytic leukemia (APL).146 HL60AR resistance to drugs and ATRA could be lowered by overexpression of dominant-negative PI3K or Akt, whereas forced expression of constitutively active Akt rendered HL60PT cells less sensitive to chemotherapeutic drugs or ATRA.88 Importantly, involvement of PI3K/Akt axis in chemoresistance has been demonstrated in AML primary cells.55

The PI3K/Akt/NF-kappaB module is also responsible for human leukemia cell line and APL blast resistance to arsenic trioxide (As2O3),119, 147, 148 another effective agent for APL treatment.149 It is interesting that PI3K/Akt signaling inhibitors decreased the intracellular glutathione content, and caused intracellular oxidation, as revealed by peroxide accumulation measurement. Cotreatment with subcytotoxic concentrations of hydrogen peroxide increased apoptosis induction by As2O3. On the other hand, the treatments did not significantly affect glutathione S-transferase pi expression and activity. These results, which highlighted glutathione as a novel target of PI3K/Akt in myeloid leukemia cells, may partially explain the selective increase of As2O3 toxicity by PI3K/Akt inhibitors,147 even though this may also be in relationship with reduced expression of antiapoptotic proteins that counteract As2O3-dependent caspase activation, as shown by our group.119

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 factor superfamily known to induce apoptosis in a wide variety of cancer, but not normal, cells.150, 151 The use of TRAIL or of agonistic antibodies to TRAIL receptors as therapeutic agents for AML has been proposed.151 A main problem, emerging from recent in vitro studies, is that AML blasts express low levels of TRAIL receptors and are therefore intrinsically resistant to this molecule.152, 153 However, expression of TRAIL receptors could be increased by cotreating AML cells with a histone deacetylase inhibitor.154 Our results have clearly established that PI3K/Akt/NF-kappaB upregulation resulted in enhanced expression of the FLICE inhibitory protein, cFLIP(L), in TRAIL-resistant HL60AR cells which, despite the expression of TRAIL receptors, did not undergo apoptosis when exposed to TRAIL.118 cFLIP(L) is an 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 downregulation of cFLIP(L) expression levels.118 Even though there is at present no clear information about upregulation of cFLIP(L) in AML cells, it may be envisaged that a combined treatment consisting of a histone acetylase inhibitor and a PI3K/Akt inhibitor, would overcome TRAIL resistance of AML blasts owing to the lack of TRAIL receptor expression concomitantly with elevated cFLIP(L) levels. In this connection, it is worth recalling here that the amount of XIAP, another member of inhibitors of apoptosis protein family which is under the control of NF-kappaB, is strongly related to prognosis in AML patients, because patients with lower levels of XIAP protein had significantly longer survival and a tendency toward longer remission duration than those with higher levels of XIAP.156 However, these conclusions have been challenged by another study.157 Whatever the case, in AML cell lines XIAP expression is at least in part under PI3K control.158

Finally, a recent study has demonstrated that both 32D cells carrying FLT-ITD and AML blasts require concurrent incubation with rapamycin (an mTOR inhibitor, see later) to undergo apoptosis in response to ATRA and the histone deacetylase inhibitor valproic acid cotreatment.159

Taken together, the aforementioned findings have provided the rationale for using pharmacological inhibitors of the PI3K/Akt network to overcome resistance to therapeutic strategies that are currently used (or that might be used in the near future) for the treatment of AML.

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Inhibition of the PI3K/Akt pathway to overcome AML therapeutic resistance

The success of tyrosine kinase inhibitors, such as erlotinib, 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 cancer patients. As activation of the PI3K/Akt axis confers therapeutic resistance, compounds that inhibit this pathway by targeting key regulatory proteins such as PI3K, Akt, mTOR or NF-kappaB have potential for new effective therapies. Either used alone or in combination with existing treatments, inhibitors of the PI3K/Akt pathway may exploit activation of the PI3K/Akt axis within AML cells to induce apoptosis and/or enhance the efficacy of other forms of treatment. We shall now discuss several pharmacological inhibitors that selectively target this signal transduction cascade. However, FLT3160 and Ras inhibitors161 will not be considered here, as they also target additional survival pathways, such as the Erk1/2 signaling network.160, 162 For the same reason, we shall also not review either VEGF163 or IGF-1 inhibitors, including the recently described NVP-AEW541.164

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 lines in which they induce apoptosis and/or increase sensitivity to chemotherapeutic drugs and TRAIL.41, 165, 166 Whereas wortmannin is a metabolite antibiotic that was first isolated from Penicillium wortmanni, LY294002 is a synthetic flavonoid derivative. Wortmannin irreversibly inhibits PI3K by covalent modification of Lys 802 of the p110 catalytic subunit, whereas LY294002 is a reversible inhibitor which competes with ATP for the ATP-binding site of PI3K.165 However, neither wortmannin nor LY294002 are entirely specific for the PI3K/Akt pathway, because wortmannin targets phospholipases C, D and A2, whereas LY294002 downregulates CK2 activity with similar potency to PI3K.41 The effect of LY294002 on CK2 appears intriguing in light of CK2-dependent Akt phosphorylation, as mentioned earlier in this article. There are several studies in which wortmannin or LY294002 have been employed to downregulate in vitro the PI3K/Akt axis of human AML cell lines or blasts. As a consequence of the treatment, cells underwent apoptosis and/or became more sensitive to chemotherapeutic drugs or TRAIL.23, 24, 58, 88, 118 An extremely interesting finding, which has emerged from these investigations, is that normal hematopoietic progenitors were less affected by PI3K inhibitors, suggesting a preferential targeting of leukemic cells.23, 24 Although the aforementioned studies demonstrated that blocking the PI3K/Akt pathway might be a valuable approach to treat AML, there are some intrinsic disadvantages with these inhibitors. Wortmannin is soluble in organic solvents that may severely limit its use in clinical trails. Currently, water-soluble wortmannin conjugates are being developed to circumvent this issue.167 As to LY294002, it has 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 xenogratfs, but some severe side effects, such as dry and scaly skin, appeared in treated mice.41 Clearly, the use of non-selective PI3K inhibitors, such as the recently described wortmannin derivative PX-866,168 is likely to be associated with undesirable side effects (e.g. hyperglycemia) because of the many important physiological targets of this lipid kinase. However, PI3K inhibitors have a hypothetical advantage in that feedback activation of the pathway, which is seen with inhibition of distal components, such as mTOR (see below), would not be observed.

Selective PI3K p110 catalytic subunit inhibitors

As the PI3K catalytic domain is highly conserved among PI3K family members,165 it is not surprising that neither wortmannin nor LY294002 discriminate among the different PI3K isozymes. IC87114 is a potent selective inhibitor of PI3K p110delta, with a IC50=0.5 muM and a >50-fold selectivity over the other class I PI3K isoforms.169 IC87114 downregulated Akt phosphorylation and almost completely blocked proliferation of AML blast cells with elevated levels of PI3K p110delta protein.71 IC87114 was as effective as LY294002 and did not affect the proliferation of normal hematopoietic precursor cells. These findings suggested that in AML patients, selective pharmacological inhibition of PI3K p110delta might offer clinical benefit and be less toxic than inhibiting all PI3K activities, even though PI3K p110delta plays a critical role in proliferation and development of the immune system. Indeed, it is well known that mice lacking functional PI3K p110delta are viable and fertile, whereas mice lacking PI3K p110alpha or beta are embryonic lethal.26

PDK-1 inhibitors

As PDK-1 is the kinase responsible for phosphorylating Akt at Thr 308, it plays a very critical role in activation of the pathway. However, PDK-1 also activates other AGC kinases that regulate cell proliferation and survival, including protein kinase C (PKC), protein kinase A and p70S6K.170 Hence, compounds that target PDK-1 will likely inhibit these other kinases. Despite the intuitive reasoning that a more selective inhibitor should have lower toxicity and cause fewer side effects, this off-target activity may be advantageous from a therapeutic standpoint but obviously complicates their development as drugs that selectively target the PI3K/Akt network, even though the use of multitargeted kinase inhibitors, such as sorafenib, has been advocated to treat cancers that are driven by several metabolic abnormabilities.171 The staurosporine derivative UCN-01, a drug now in phase II clinical trials, has been shown to potently inhibit PDK-1 in vitro and in vivo, by forming a complex with the kinase domain of PDK-1.172, 173 UCN-01 is capable of interacting synergistically with the farnesyltransferase inhibitor L744832, thereby increasing apoptosis of HL60 cells and AML blasts.102 Moreover, a recent pilot clinical trial of cytarabine in combination with UCN-01 in patients with relapsed AML, has shown a decline in Akt kinase activation which was accompanied by a decrease in checkpoint kinase 1 (Chk1) phosphorylation, an activation of JNK, and reduction in absolute AML blast counts.174 These findings offer a rationale for the cytotoxic action of this combination therapy for AML treatment. Nevertheless, UCN-01 also inhibits PKC-alpha, -beta and -italic gamma, as well as Chk1, so that its specificity of action remains to be better defined.175

Selective Akt inhibitors

Phosphatidylinositol ether analogues
 

Most small molecular weight kinase inhibitors are ATP mimics. The ATP cleft is strongly conserved between kinases, so ATP-competitive inhibitors tend to lack specificity and to affect groups of related kinases. An alternative approach to inhibiting Akt is to target its PH domain and interfere with PtdIns (3,4,5)P3 binding and membrane translocation. Phosphatidylinositol ether analogues (PIAs) have been designed to inhibit this interaction. The rationale behind the synthesis of these molecules is that they cannot be phosphorylated by PI3K on the 3-position of the myo-inositol ring. Indeed, they act as competitors for Akt activation at the plasma membrane, hence they behave as inhibitors downstream of PI3K and PDK-1. As outlined above, a conceptual difference in the development of PIAs to inhibit Akt is that targeting the PH domain should minimize the lack of specificity observed with compounds that target the ATP-binding domain of Akt.176 Indeed, the Akt isoform PH domains are only about 30% identical to PH domains in other proteins.177 Importantly, PIAs selectively induced apoptosis in several cancer cell lines that have high levels of Akt phoshorylation and were only modestly active in tumor cells displaying low levels of phosphorylated Akt.178 Moreover, one of these compounds, PX-316, displayed in vivo antitumor activity against human MCF-7 breast cancer and HT-29 colon cancer xenografts in mice. Both these cell lines have an upregulated PI3K/Akt network. Remarkably, PX-316 formulated in 20% hydroxypropyl-beta-cyclodextrin for intravenous administration was well tolerated in mice and rats with no hemolysis and no hematological toxicity.179

In keeping with these findings and by exploiting the experimental system consisting of HL60AR cells, we demonstrated that one of these PIAs (1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate) was able to restore sensitivity to chemotherapeutic drugs, ATRA and TRAIL.180 Furthermore, we have tested two novel PIAs with improved metabolic stability and anticancer potential, D-3-deoxy-2-O-methyl-myo-inositol 1-((R)-2-methoxy-3-(octadecyloxy)propyl hydrogen phosphate and D-2,3-dideoxy-myo-inositol 1-((R)-2-methoxy-3-(octadecyloxy)propyl hydrogen phosphate (see compounds PIA5 and PIA6 in Castillo et al.178) on HL60AR cells. They were able to markedly increase sensitivity of HL60AR cells to etoposide or cytarabine at a concentration (5 muM), which was not toxic to human cord blood CD34+ hematopoietic precursor cells.181 Key issues for the development of lipid-based Akt inhibitors are oral bioavailability and hemolysis, a side efffect that may determine which of these compounds will eventually emerge as a front runner.176

Other Akt inhibitors
 

Over the past 3 years, several other Akt inhibitors have been described, including perifosine (a novel orally bioavailable alkylphospholipid that inhibits Akt phosphorylation by preventing its membrane localization, possibly through interaction with the PH domain, see Kondapaka et al.182), deguelin (a naturally occurring plant rotenoid, whose mechanism of action is unclear, see Chun et al.183), allosteric Akt kinase inhibitors that are isozyme specific and require the PH domain of Akt,184, 185 the indazole-pyridine compound A-443654,186 API-2/tricribine187 and a cell-permeable neutralizing single-chain antibody to Akt.188 Of all the above inhibitors, only deguelin and perifosine have been tested on AML cell lines. At 10–100 nM concentration, deguelin downregulated Akt phosphorylation of U937 leukemia cells and markedly increased their sensitivity to etoposide or cytarabine. A 10 nM concentration of deguelin did not negatively affect the survival rate of human cord blood CD34+ cells. Moreover, deguelin was less toxic than wortmannin on erythropoietin (EPO)- and SCF-induced erythropoiesis from CD34+ progenitor cells.189 As to perifosine, it has been shown that it synergized with a histone acetylase inhibitor to induce a decrease of cell proliferation and an increase in apoptosis of HL60 cells,190 even if it was not demonstrated that the drug actually targeted Akt in this experimental system. Nevertheless, potential efficacy of perifosine in AML with activated PI3K/Akt signaling, is indicated by the results of a recent study in which perifosine induced significant apoptosis in multiple myeloma cell lines and patient multiple myeloma cells, characterized by upregulation of PI3K/Akt network.191 Moreover, perifosine augmented dexamethasone, doxorubicin, melphalan and bortezomib-induced multiple myeloma cell cytotoxicity and demonstrated significant antitumor activity in a human plasmacytoma mouse model. In this investigation, the perifosine IC50 in vitro effective on neoplastic plasma cells (1.5–15 muM) was within the range of plasma concentrations achieved in vivo (approx16 muM) during a phase I study of perifosine in solid tumors.192 Thus, a phase II trial of perifosine on multiple myeloma patients has very recently begun.

The allosteric Akt kinase inhibitors are particularly interesting, because they can target specifically Akt1, Akt2 or both Akt1 and Akt2. Therefore, at least in theory, the use of isozyme-selective Akt inhibitors might result in less pronounced unpleasant side effects. For example, as outlined above, studies on knockout mice have revealed that Akt2 plays a very important role in insulin-mediated glucose homeostasis, as also demonstrated by the fact that this Akt isoform is the most abundantly expressed in glucose-sensitive tissues and organs.193 Therefore, it would be very important to define if in AML blasts both Akt1 and Akt2 are upregulated, or if only one of the two isoforms is involved in conveying survival signals. However, according to the available evidence, maximal induction of apoptosis by isozyme-selective Akt inhibitors in several cancer cell lines was achieved with the Akt1/Akt2 dual inhibitor.185 These isoform-specific inhibitors were only modestly active in inducing apoptosis in tumor cells, but synergized with chemotherapeutic agents to induce apoptosis. Moreover, these compounds have poor solubility and pharmacokinetics properties that precluded their evaluation in animal tumor model.185 Overall, Akt inhibition would be expected to inhibit most, if not all, of Akt substrates. Because we are far away from the identification of all Akt substrates and critical substrates can vary with cell type, inhibition of individual downstream components of PI3K/Akt pathway may 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 of potential greater toxicity.

HSP-90 inhibitors

Heat-shock protein-90 is currently receiving consideration as a potential anticancer drug target. The ability of HSP-90 to stabilize client proteins is inhibited by the benzoquinone ansamycin antibiotic geldanamycin and its derivatives, that occupy the ATP-binding site on HSP-90 and promote protein degradation via the proteasome pathway.194 Differences between chaperoning complexes of neoplastic and healthy cells make HSP-90 an attracting target for anticancer treatment. Indeed, in neoplastic cells, HSP-90 forms multimolecular complexes with both high ATP-ase activity and a high affinity for the geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG), whereas in normal cells it exists as a latent form which displays low ATPase activity and low affinity for 17-AAG.195 These findings have provided a rationale for the enhanced accumulation and selective cytotoxicity of 17-AAG in cancer cells.194 Several reports have indicated that HSP-90 inhibitors including 17-AAG, either used alone or in combination with other treatments, selectively induced apoptosis in leukemic cells harboring activating mutations of FLT3196, 197, 198 or c-Kit.199 Akt dephosphorylation was observed in one of these studies,196 along with downregulation of other FTL3 downstream effectors (STAT5a, Erk1/2). However, as for UCN-01, it should be pointed out that 17-AAG, by targeting HSP-90, also inhibits other kinases including Chk1,200 so that it cannot be considered selective for the PI3K/Akt pathway.

Mammalian target of rapamycin inhibitors

An alternative target to either PI3K or Akt could be represented by kinases located downstream of Akt, such as mTOR. Mammalian target of rapamycin is particularly interesting because, besides its important role in cell metabolism, it has been recently demonstrated to behave as a critical Akt activator by forming a complex with Rictor.36 The Rictor/mTOR complex directly phosphorylated Akt on Ser 473 in vitro and facilitated Thr 308 phoshorylation. Therefore, the Rictor/mTOR complex might be the much sought after PDK-2, which phosphorylates Akt on Ser 473 in response to growth factor stimulation.201 Mammalian target of rapamycin inhibitors are the most developed class of compounds that target the PI3K/Akt pathway and include: rapamycin, CCI-779, RAD001 and the phosphorous-containing derivative AP23573.202 Rapamycin is a macrocyclic lactone antibiotic, produced by Streptomyces hygroscopicus, which potently inhibits the growth of cancer cell lines and induces apoptosis. It does not directly inhibit mTOR, but rather binds to its immunophilin, FK506-binding protein 12 (FKBP12). Then, rapamycin/FKBP12 complex binds to mTOR complexed with Raptor (Figure 2) and inhibits downstream signaling events.202 Rapamycin has been approved by FDA as an immunosuppressant, and in phase I/II clinical trials has shown activity against many types of cancer.139 It has, however, two disadvantages: poor solubility and chemical stability. For this reason, ester analogues of rapamycin with improved aqueous stability and solubility have been synthesized.203 CCI-779 has been designed for intravenous injection, whereas RAD001 is available for oral administration. A phase II clinical trial with CCI-779 in patients with metastatic renal carcinoma has already been completed and the drug is now in phase III clinical trials. RAD001 (which is approved in Europe as an immunosuppressant agent in solid organ trasplantation) and AP23573167 are currently undergoing phase I/II clinical trials as antitumor drugs.

Rapamycin failed to reverse drug resistance in HL60 cells with upregulated PI3K/Akt signaling.88, 145 In contrast, it has been reported that RAD001 was capable of sensitizing U937 leukemia cells to cytarabine.23 The reason for these conflicting findings is unclear. It might be due to the different cell types and/or experimental conditions employed for these investigations, nevertheless it could also be related to rapamycin resistance (see later). Rapamycin had only a modest effect on primary AML cell survival in liquid culture; however, it markedly impaired AML blast clonogenicity while sparing normal hematopoietic precursors. Moreover, it was able to induce a significant and rapid clinical response in vivo in four of nine patients with either refractory/relapsed de novo or secondary AML.204 Accordingly, 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) cultures the effect was more pronounced. Moreover, rapamycin cytotoxicity in short term cultures could be dramatically increased by co-treatment with etoposide.94 Importantly, etoposide toxicity on CD34+ cells from healthy donors was not enhanced by addition of rapamycin.

A synergism has also been reported for a combination treatment consisting of rapamycin and UCN-01.205 This combination treatment resulted in marked potentiation of apoptosis in U937 cells that was accompanied by a decrease in the levels of Mcl-1. Mcl-1 protein expression levels are related with resistance of human leukemias to a variety of chemotherapeutic 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 cytotoxic effect on HL60 leukemia cells is that constituted of rapamycin and the cell permeable glycolytic inhibitor 3-bromo-2-oxoproprionate-1-propyl ester.209 The efficacy of this combination treatment conceivably depends on the fact that in cancer cells, which are very hypoxic, the PI3K/Akt/mTOR axis elicits the long-known 'Warburg effect' where glucose uptake, glycolysis and lactate production are accelerated without any change in oxygen consumption.210 Enhanced glycolysis could be related to overexpression of hypoxia-inducible factor (HIF)-1alpha. Mammalian target of rapamycin upregulates HIF-1alpha, primarily by increasing the rate of HIF-1alpha protein translation.211 Interestingly, VEGF expression is increased by HIF-1alpha.212

The above results seem to indicate that rapamycin or its derivative, either alone or in combination, might be very promising drugs for the treatment of AML. However, in light of recent findings a caveat is necessary, because it is emerging that mTOR also mediates suppression of PI3K activation. Indeed, mTOR phosphorylates insulin receptor substrate-1, resulting in its proteosomal degradation and downregulation of IGF-1-evoked signaling to PI3K/Akt.213, 214 Even if there is no evidence of the existence of a such a negative feedback loop in AML cells, it is clear that mTOR inhibition could result in upregulation of growth factor-dependent PI3K/Akt signaling. However, the occurrence of this inhibitory mechanism has been very recently demonstrated in multiple myeloma cells, in which either rapamycin or CCI-779 treatment resulted in Akt activation in vivo, suggesting that such feedback also takes place in hematopoietic cells.215 Although the role of serum IGF-1 in supporting proliferation and survival of myeloma cells both in vitro and in vivo is well established,216 its involvement in AML remains to be demonstrated, but could not be completely ruled out. These findings raise a caution about the indiscriminant use of rapamycin in cancer therapy.

In principle, therefore, therapeutic approaches that simultaneously target both PI3K/Akt and mTOR/Raptor complex, like the combination rapamycin/UCN-01, may ultimately prove more efficacious. Moreover, another potential weakness of rapamycin is represented by the fact that this drug could be exported from cells through ATP-binding cassette (ABC) type transporters that mediate multidrug resistance such as 170-kDa P-glycoprotein,217 and AML cells are known to express several of these transporters.218 Rapamycin extrusion by ABC transporters could also account for marked differences in the drug concentration (range: from less than 1 nM to more than 100 nM) necessary to achieve IC50 on AML blast clonogenic assay and could also explain why some patients did not respond at all to rapamycin treatment.204 However, other mechanisms of rapamycin resistance have been identified.135

NF-kappaB inhibitors

As underlined above, I-kappaB/NF-kappaB is upregulated in AML blasts leading to the expression of antiapoptotic c-IAP2, whereas unstimulated human hematopoietic progenitor cells do not express NF-kappaB.120, 219 Importantly, molecular genetic studies using a dominant-negative allele of I-kappaB demonstrated that inhibition of NF-kappaB contributed to apoptotic cell death of LSCs.220 These observations have provided a rationale for the use of NF-kappaB inhibitors as potential therapeutic agents against AML. There exists an extremely wide variety of natural and synthetic NF-kappaB inhibitors.221 For example, curcumin, a yellow coloring agent from turmeric (Curcuma longa rhizomes), commonly used as a spice, is well documented for its medicinal properties in Indian and Chinese systems of medicine. Several reports have highlighted that curcumin causes apoptosis in human leukemia cell lines and downregulates NF-kappaB activity.222, 223 Capsaicin, a homovanillic acid derivative found in pungent fruits, is another natural NF-kappaB inhibitor which has been shown to induce growth inhibition and apoptosis of human myeloid leukemic cell lines in vivo and in vitro.224 Two other natural NF-kappaB inhibitors have been recently described and demonstrated to possess in vitro pharmacological activity against primary AML cells. Resveratrol, an edible polyphenolic stilbene found in the skin of red grapes and various other fruits, inhibited NF-kappaB activity of AML cell lines and blasts. NF-kappaB inhibition correlated with increased apoptosis.225 The other natural compound capable of downregulating NF-kappaB in AML blasts is indole-3-carbinol, which is found in Brassica species vegetables (i.e., cabbage, cauliflower, brussels sprout). In this case, it was shown that several of antiapoptotic genes, whose expression is controlled by NF-kappaB (XIAP, cIAP-1, cIAP-2, cFLIP, TRAF-1), were suppressed by indole-3-carbinol.226

As to synthetic inhibitors, SN50, a peptide which blocks nuclear import of NF-kappaB,227 decreased TRAIL or As2O3 resistance of HL60AR cells, as shown by our laboratory.118, 119 A recent study has highlighted that AS602868, a small molecule which selectively targets IKKbeta, induced apoptosis of AML blasts and also potentiated the apoptotic response induced by chemotherapeutic drugs currently used for the treatment of AML, such as doxorubicin, cytarabine and etoposide.121 BAY-11–7082 is yet another synthetic NF-kappaB inhibitor which caused apoptosis in human leukemic cell lines.228

All these studies have validated NF-kappaB as a promising therapeutic target downstream of PI3K/Akt in AML. It may also be worth mentioning here that bortezomib, an indirect NF-kappaB inhibitor which targets the proteasome,229 has been approved for treatment of multiple myeloma230 and is currently under clinical evaluation for other hematological malignancies, including AML.231 Interestingly, bortezomib induced apoptosis of bone marrow monuclear myeloid (BMMM) cells from patients with high-risk MDS, a preneoplastic condition which frequently develops into overt AML, and is characterized by a progressive increase in bone marrow blasts with reduced apoptotic capacities.232 In this connection, our laboratory has very recently shown that BMMM cells from high-risk MDS patients frequently display high levels of phosphorylated Akt, which might be responsible for NF-kappaB upregulation.233

However, a cautionary note is required here. Although NF-kappaB activation in most cases is harmful, there is evidence that NF-kappaB upregulation has also beneficial effects. Indeed, NF-kappaB is required for normal functioning of the immune system, for hematopoiesis, and could even promote apoptosis,234 for example by positively regulating the expression of TRAIL DR5 receptor.235 Therefore, NF-kappaB inhibitors should be tested with caution in view of the dual nature of this transcription factor.

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Concluding remarks and future directions

A growing excitement surrounds the development of signal transduction modulators as new powerful agents for treating malignant disorders. There is no doubt that the activation of the PI3K/Akt pathway confers resistance to therapeutic treatments of various types of cancer both in vivo and in vitro, including AML. This finding has driven the frantic development of compounds directed against components in the pathway. Nevertheless, a fundamental issue that still awaits answering is: will inhibition of this signaling network negatively affect human disorders without deleterious side effects, such as perturbations of glucose homeostasis? In other words, is there a therapeutic window when such an ubiquitous and fundamental pathway is targeted? In vitro data show that inhibitors are preferentially cytotoxic for tumor cells that exhibit increased PI3K/Akt activation, suggesting that death of cancer cells without death of healthy cells is possible. Perhaps a basis for a potential therapeutic window in vivo can be attributed to increased reliance on pathways promoting cellular survival by cancer cells exposed to forms of stress (e.g. chemotherapy) that are known for activating PI3K/Akt. In addition, tumor cells might be more sensitive than normal cells to inhibition of this network because they often grow in harsh conditions deprived of nutrients and would therefore be highly reliant on a signaling pathway which has been upregulated during disease progression for their survival (the so-called 'addiction hypothesis').236 Thus, even a partial inhibition of this pathway might be sufficient to negatively affect neoplastic survival and proliferation while sparing normal cells. Obviously, it will be of the outmost importance the selection of patients with molecular dependence on the pathway.

How could the biological effectiveness of PI3K/Akt pathway inhibitor be measured in vivo? At variance with patients with solid tumors, the evaluation of the efficacy of the therapeutic treatment should be more easily feasible in AML patients, using either peripheral blood or bone marrow samples. Existing assays that could be used to determine pathway inhibition include immunohistochemistry with phosphospecific or native antibodies recognizing levels of active or total protein, respectively,54, 233, 237 kinase assays,57 and/or flow cytometry using the same antibodies whenever possible.56, 57, 238 Flow cytometry appears particularly well suited for evaluation of AML patients, as it offers obvious advantages over the other techniques (especially Western blot and kinase assays), including quickness, a lower number of cells required to perform the assay, and the possibility of identifying different subclones in the leukemic populations by coimmunostaining with multiple antibodies to surface antigens, even when the percentage of blast cells is usually low such as in some AML subgroups (FAB M2, M4, M5).57, 239, 240 The use of this technique is now greatly facilitated by the availability of fluorochrome-conjugated primary antibodies directed to phosphoproteins, including Akt, and by the possibility of performing immunostaining in the whole blood.241 Moreover, flow cytometry would be extremely well suited for the analysis of the effects of treatment on the rare LSCs, the real target for AML eradication.

The side effects reported so far in patients treated with inhibitors of this pathway (CCI-779, perifosine, UCN-01) include thrombocytopenia, anemia, liver disfunction, hypocalcemia, maculopapular rash, mucositis, hyperglycemia, nausea, vomiting and diarrhea.202, 242, 243 The occurrence of hyperglycemia raises the issue of impaired glucose tolerance and type II 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 with indazole-pyridine Akt inhibitors.186 Even though blood glucose levels were normal, the increase in serum insulin is consistent with a homeostatic response, where the animals secrete more insulin to maintain blood glucose concentrations and oppose insulin resistance. This is also what has been observed in Akt2 knockout mice.33 When young, these animals have normal blood glucose concentrations but increased plasma insulin. Only when older, after pancreatic islet beta-cell failure, do the animals lose their ability to maintain normal blood glucose levels. This indicates that PI3K/Akt inhibitors might induce type 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 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? Obviously, shorter exposures in patients might be associated with less toxicity. However, we do not know whether or not 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.

As to the occurrence of thrombocytopenia and anemia, this might suggest detrimental effects of the inhibitors on hematopoiesis. This may appear surprising, because, according to the literature, phosphorylated Akt is extremely low or absent in normal bone marrow.54, 233, 237 Furthermore, several investigations have shown that PI3K/Akt inhibitors are much less toxic for normal human hematopoietic precursor cells than for AML blasts.23, 71, 121, 204 However, it should not been overlooked that many cytokines activate PI3K/Akt signaling which therefore would seem to play an important role in erythropoiesis, myelopoiesis and thrombocytopoiesis.244, 245, 246, 247 Clearly, these results have been obtained in vitro and they might not reflect what happens in vivo. In addition, also in this field the findings are conflicting, as exemplified by a recent paper which suggests that EPO downregulates PI3K/Akt axis during erythropoiesis through upregulation of phosphatidylinositol 4-phosphatase II,248 whereas other reports have suggested a key role for PI3K/Akt in EPO-mediated erythroid differentiation.245, 249, 250

One of the challenges that lie ahead is understanding whether PI3K/Akt inhibitors will be effective in vivo when employed alone or will require to be combined with other treatments, such as chemotherapy. Conceivably, a combination therapy would allow the use of lower dosage of signal transduction modulators giving maximum efficacy and minimum side effects, as substantial evidence is accumulating for synergy between kinase inhibition and classical chemotherapy.

Finally, what component would be the best target in such a heavily branched signaling network? Would it be preferable to target single components of the branches further downstream of PI3K/Akt, such as mTOR or NF-kappaB, that are more exclusively involved in cell growth, proliferation and survival? Or would 'cocktails' of drugs affecting multiple steps of the pathway to be an even more effective form of therapy? Indeed, the PI3K/Akt pathway bifurcates and integrates with other signaling networks as the signal is propagated. The pathway could therefore be more globally, and thereby effectively, inhibited when also targeting PI3K or Akt. At least in theory, this kind of inhibition might also be less susceptible to the complicating and still largely ill-defined effects of feedback loops than the inhibition of single branches downstream. However, this efficacy would presumably be balanced by potential for higher toxicity and a narrower therapeutic index.

The last several years have witnessed major progress towards the goal of translating our growing understanding of the molecular basis of cancer into drugs with improved therapeutic activity and selectivity. Tremendous advances have been made but significant obstacles remain. Additional work addressing all the aforementioned issues is needed to determine if the potential of PI3K/Akt inhibitors may be fully realized in AML treatment. However, the continuing efforts to develop specific, high-affinity PI3K/Akt inhibitors will surely yield new effective drugs. Intelligent clinical trial design, coupled with rigorous scientific evaluation, may herald entering a new era in which signal transduction modulators could significantly improve the outcome of AML.

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Acknowledgements

This work was supported by grants from: Italian MIUR FIRB 2001 and COFIN 2005, Associazione Italiana Ricerca sul Cancro (AIRC Regional Grants), CARISBO Foundation.

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