The PI3K/PKB Signaling Module in Normal and Malignant Hematopoiesis

Hematopoiesis is a complex series of events resulting in the formation of mature blood cells. This process is regulated by cytokines at various levels, including self-renewal, proliferation, and differentiation. Upon binding of cytokines to their cognate receptors, the activity of intracellular signal transduction pathways is regulated, leading to modulation of gene expression. Although our appreciation of the transcriptional regulators of hematopoiesis has developed considerably, until recently, the roles of specific intracellular signal transduction pathways were largely unknown. An important mediator of cytokine signaling implicated in regulation of hematopoiesis is the Phosphatidylinositol-3-Kinase (PI3K) / Protein Kinase B (PKB/c-Akt) signaling module (Figure 1). The PI3K family consists of three distinct subclasses of which, to date, only the class I isoforms have been implicated in regulation of hematopoiesis. Four distinct catalytic class I isoforms have been identified; p110, p110, p110and p110(reviewed by Vanhaesebroeck et al., 2001) These isoforms are predominantly activated by protein tyrosine kinases and form heterodimers with a group a regulatory adapter molecules, including p85, p85, p50 p55, p55and p101(reviewed by Vanhaesebroeck et al., 2001). The most important substrate for these Class I PI3Ks is phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) which can be phosphorylated at the D3 position of the inositol ring upon extracellular stimulation, resulting in the formation of phosphatidylinositol 3,4,5 trisphosphate (PI(3,4,5)P3) (reviewed by Hawkins et al., 2006). PI(3,4,5)P3 subsequently serves as an anchor for pleckstrin homology (PH) domain-containing proteins, such as Protein Kinase B (PKB/ c-akt) (Burgering & Coffer, 1995). Activation of PKB requires phosphorylation on both Thr308, in the activation loop, by phosphoinositide-dependent kinase 1 (PDK1) and Ser473, within the carboxyl-terminal hydrophobic motif, by the MTORC2 complex that consists of multiple proteins, including Mammalian Target of Rapamycin (mTOR) and Rictor (Sarbassov et al., 2005). PKB itself subsequently regulates the activity of multiple downstream effectors, including the serine/threonine kinase Glycogen Synthase Kinase-3 (GSK-3) (Cross et al., 1995), members of the FoxO subfamily of forkhead transcription factors FoxO1, FoxO3, and FoxO4 (Brunet et al., 1999; Kops et al., 1999) and the serine/threonine kinase mammalian target of rapamycin (mTOR) as part of the MTORC1 complex, which also includes the regulatory


Introduction
Hematopoiesis is a complex series of events resulting in the formation of mature blood cells.This process is regulated by cytokines at various levels, including self-renewal, proliferation, and differentiation.Upon binding of cytokines to their cognate receptors, the activity of intracellular signal transduction pathways is regulated, leading to modulation of gene expression.Although our appreciation of the transcriptional regulators of hematopoiesis has developed considerably, until recently, the roles of specific intracellular signal transduction pathways were largely unknown.An important mediator of cytokine signaling implicated in regulation of hematopoiesis is the Phosphatidylinositol-3-Kinase (PI3K) / Protein Kinase B (PKB/c-Akt) signaling module (Figure 1).The PI3K family consists of three distinct subclasses of which, to date, only the class I isoforms have been implicated in regulation of hematopoiesis.Four distinct catalytic class I isoforms have been identified; p110, p110, p110and p110(reviewed by Vanhaesebroeck et al., 2001) These isoforms are predominantly activated by protein tyrosine kinases and form heterodimers with a group a regulatory adapter molecules, including p85, p85, p50 p55, p55and p101(reviewed by Vanhaesebroeck et al., 2001).The most important substrate for these Class I PI3Ks is phosphatidylinositol 4,5 bisphosphate (PI(4,5)P 2 ) which can be phosphorylated at the D3 position of the inositol ring upon extracellular stimulation, resulting in the formation of phosphatidylinositol 3,4,5 trisphosphate (PI(3,4,5)P 3 ) (reviewed by Hawkins et al., 2006).PI(3,4,5)P 3 subsequently serves as an anchor for pleckstrin homology (PH) domain-containing proteins, such as Protein Kinase B (PKB/ c-akt) (Burgering & Coffer, 1995).Activation of PKB requires phosphorylation on both Thr 308 , in the activation loop, by phosphoinositide-dependent kinase 1 (PDK1) and Ser 473 , within the carboxyl-terminal hydrophobic motif, by the MTORC2 complex that consists of multiple proteins, including Mammalian Target of Rapamycin (mTOR) and Rictor (Sarbassov et al., 2005).PKB itself subsequently regulates the activity of multiple downstream effectors, including the serine/threonine kinase Glycogen Synthase Kinase-3 (GSK-3) (Cross et al., 1995), members of the FoxO subfamily of forkhead transcription factors FoxO1, FoxO3, and FoxO4 (Brunet et al., 1999;Kops et al., 1999) and the serine/threonine kinase mammalian target of rapamycin (mTOR) as part of the MTORC1 complex, which also includes the regulatory associated protein of mTOR (Raptor).In contrast to GSK-3 and the FoxO transcription factors that are inhibitory phosphorylated by PKB, activation of mTOR is positively regulated (Nave et al., 1999;Inoki et al., 2002).It has been demonstrated that PKB can inhibit the GTPase activating protein Tuberous sclerosis protein 2 (TSC2)/TSC1 complex, resulting in accumulation of GTP-bound Rheb and subsequent activation of mTOR (Inoki et al., 2002).Fig. 1.Schematic representation of the PI3K/PKB signaling module.Activation of PI3K by receptor stimulation results in the production of PtdIns(3,4,5)P 3 at the plasma membrane.PKB subsequently translocates to the plasma membrane where it is phopshorylated by PDK1 and the mTORC2 complex.Upon phosphorylation, PKB is released into the cytoplasm where it can both inhibitory phosphorylate multiple substrates, including FoxO transcription factors and GSK-3 and induce the activity of other substrates such as mTOR as part of the mTORC1 complex.Negative regulators of the PI3K/PKB signaling module include PTEN, SHIP1 and Ins(1,3,4,5)P4.
While cytokines and growth factors positively induce PI3K activity, its activity can also be inhibited by SH2-containing inositol-5'-phosphatase 1 (SHIP1) (Damen et al., 1996), a protein predominantly expressed in hematopoietic cells (Liu et al., 1998), that hydrolyzes PIP 3 to generate PI(3,4)P 2 (Damen et al., 1996).Similarly, Phosphate and Tensin Homologue (PTEN) (Maehama & Dixon, 1998), a ubiquitously expressed tumor suppressor protein, can least partially due to activation of the PI3K/PKB signaling module.Taken together, these studies suggest that correct temporal regulation of PI3K activity is critical for both HSC maintenance and regulation of lineage development.

PKB
PKB, an important effector of PI3K signaling, has been demonstrated to play an important role in regulation of cell survival and proliferation in a variety of systems (reviewed by Manning & Cantley, 2007).Three highly homologous PKB isoforms have been described to be expressed in mammalian cells; PKB, PKB, and PKB.Analysis of HSCs derived from PKB/PKB double-knockout mice revealed that PKB plays an important role in maintenance of long-term repopulating HSCs.These PKB/PKB double-deficient HSCs were found to persist in the G 0 phase of the cell cycle, suggesting that the long-term functional defects observed in these mice were caused by enhanced quiescence (Juntilla et al., 2010).In contrast, loss of only one of the isoforms only minimally affected HSCs (Juntilla et al., 2010).In addition, analysis of mice deficient for both PKB and PKB revealed that the generation of marginal zone and B1 B cells and the survival of mature follicular B cells highly depend on the combined expression of PKB and PKB.Again no significant differences could be observed in mice deficient for the single isoforms (Calamito et al., 2010).In addition, ectopic expression of constitutively active PKB in mouse HSCs conversely resulted in transient expansion and increased cycling of HSCs, followed by apoptosis and expansion of immature progenitors in BM and spleen, which was also associated with impaired engraftment (Kharas et al., 2010), again demonstrating the importance of PKB in HSC maintenance.Utilizing an ex vivo human granulocyte differentiation system and a mouse transplantation model, it has recently been demonstrated that PKB not only plays a role in expansion of hematopoietic progenitors, but also has an important function in regulation of cell fate decisions during hematopoietic lineage commitment (Buitenhuis et al., 2008).High PKB activity was found to promote neutrophil and monocyte development and to inhibit B lymphocyte development, while conversely reduction of PKB activity is required to induce optimal eosinophil differentiation (Buitenhuis et al., 2008).In addition, PKB plays an important role in regulation of proliferation and survival of dendritic cell (DC) progenitors, but not maturation (van de Laar et al., 2010).Transplantion of mouse bone marrow cells ectopically expressing constitutively active PKB was sufficient to induce a myeloproliferative disease in most mice, characterized by extramedullary hematopoiesis in liver and spleen.In the majority of those mice, lymphoblastic thymic T cell lymphoma could also be observed.In addition, an undifferentiated AML developed in those mice that did not develop a myeloproliferative disease (Kharas et al., 2010).

Downstream effectors of PKB
To understand the molecular mechanisms underlying PKB mediated regulation of hematopoiesis, the roles of its downstream effectors in hematopoiesis have been investigated.FoxO transcription factors are known to play an important role in regulation of proliferation and survival of various cell types (reviewed by Birkenkamp & Coffer, 2003).Although proliferation and differentiation of hematopoietic progenitors appears not to be affected in FoxO3 deficient mice, competitive repopulation experiments revealed that deletion of FoxO3 is sufficient to impair long-term reconstitution (Miyamoto et al., 2007).In addition, in aging mice, the frequency of HSCs was increased compared to wild type littermate controls (Miyamoto et al., 2007) and neutrophilia developed upon myelosuppressive stress conditions (Miyamoto et al., 2007).In contrast to FoxO3 deficient mice in which neutrophilia only occurred after myelosuppression while aging, conditional deletion of FoxO1, 3, and 4 in the adult hematopoietic system, was sufficient to increase the levels of myeloid cells and decrease the number of peripheral blood lymphocytes under normal conditions.In time, these mice developed leukocytosis characterized by a relative neutrophilia and lymphopenia (Tothova et al., 2007).In addition, an initial expansion of HSCs has been observed in these mice which correlated with an HSC-specific up-regulation of Cyclin D2 and down-regulation of Cyclin G2, p130/Rb, p27, and p21 (Tothova et al., 2007).Furthermore, a defective long-term repopulating capacity of bone marrow cells was observed, which could be explained by the reduction in HSC numbers that followed the initial expansion (Tothova et al., 2007).Although deletion of FoxO3 alone was not sufficient to improve myeloid development, ectopic expression of a constitutively active, non-phosphorylatable, FoxO3 mutant in mouse hematopoietic progenitors did result in a decrease in the formation of both myeloid and erythroid colonies (Engstrom et al., 2003), suggesting that FoxO3 does plays an important role in lineage development.Modulation of the activity of the PI3K signaling pathway has been observed to alter the level of reactive oxygen species (ROS).While ROS levels are reduced in PKB/ deficient mice (Juntilla et al., 2010), increased levels have been observed in mice deficient for FoxO (Miyamoto et al., 2007).Increasing ROS levels in PKB/ deficient mice was sufficient to rescue differentiation defects, but not impaired long-term hematopoiesis (Juntilla et al., 2010).Restoring the ROS levels in FoxO deficient mice by in vivo treatment with an antioxidative agent N-acetyl-L-cysteine was sufficient to abrogate the enhanced levels of proliferation and apoptosis in FoxO deficient HSCs and to restore the reduced colony forming ability of these cells (Tothova et al., 2007).These studies demonstrate that correct regulation of ROS by FoxO transcription factors is essential for normal hematopoiesis.Recent findings have demonstrated that correct regulation of the activity of GSK-3, another downstream effector of PKB, is also essential for maintenance of hematopoietic stem cell homeostasis.A reduction in long-term, but not short-term repopulating HSCs has, for example, been observed in GSK3 deficient mice (Huang et al., 2009).In addition, disruption of GSK-3 activity in mice with a pharmacological inhibitor or shRNAs has been shown to transiently induce expansion of both hematopoietic stem and progenitor cells followed by exhaustion of long-term repopulation HSCs (Trowbridge et al., 2006;Huang et al., 2009).In addition, since GSK-3 has been demonstrated to inhibit mTOR activity by phosphorylation and activation of TSC1/2 (Inoki et al., 2006) and the level of phosphorylated S6 was enhanced in cells with reduced GSK-3 levels, mice were treated with rapamycin.Rapamycin induced the number of LSK cells when GSK3 was depleted, but not in un-manipulated cells, suggesting that mTOR is an important effector of GSK-3 in regulation of HSC numbers (Huang et al., 2009) In addition to the observed expansion of HSCs in mice treated with a GSK-3 inhibitor, the recovery of neutrophil and megakaryocyte numbers after transplantation was accelerated in these mice, resulting in improved survival of the recipients (Trowbridge et al., 2006).In addition, ex vivo experiments revealed that GSK-3 can enhance eosinophil differentiation and inhibit neutrophil development (Buitenhuis et al., 2008).C/EBP, a key regulator of hematopoiesis, has been demonstrated to be an important mediator of PKB/GSK-3 signaling in regulation of granulocyte development (Buitenhuis et al., 2008).
A third, important mediator of PI3K/PKB signaling is mTOR.Conditional deletion of TSC1 in mice, resulting in activation of mTOR, has been demonstrated to enhance the percentage of cycling HSCs and to reduce the self-renewal capacity of HSCs in serial transplantation assays (Chen et al., 2008).In addition, a reduction in the number of granulocytes and lymphocytes has been observed in those mice (Chen et al., 2008).As described above, activation of the PI3K signaling pathway by conditional deletion of PTEN in adult murine HSCs resulted in an initial expansion followed by exhaustion of LT-HSCs.Inhibition of mTOR in murine HSCs deficient for PTEN with Rapamycin was sufficient to revert this phenotype, again suggesting that mTORC1 signaling plays an important role in proliferation of HSCs (Yilmaz et al., 2006).A role for mTOR in progenitor expansion has been demonstrated utilizing an ex vivo human granulocyte differentiation system (Geest et al., 2009).In contrast to inhibition of PKB activity which not only affects progenitor expansion but also alters lineage development (Buitenhuis et al., 2008), inhibition of mTOR activity with Rapamycin only reduced the expansion of hematopoietic progenitors, during both eosinophil and neutrophil differentiation, without altering levels of apoptosis or maturation (Geest et al., 2009).Similarly, inhibition of mTOR reduced the number of interstitial DCs and Langerhans cells in in vitro experiments (van de Laar et al., 2010).In contrast to granulocyte development, treatment with rapamycin appears not only to affect proliferation during megakaryocyte (MK) development, but also appears to delay the generation of pro-platelet MKs (Raslova et al., 2006).Similar to FOXO transcription factors, TSC1 also appears to be involved in regulation of ROS levels in HSCs.Elevated levels of ROS have been observed in TSC1 deficient mice.In vivo treatment of those mice with a ROS antagonist restored HSC numbers and function (Chen et al., 2008), suggesting that TSC1 regulates HSC numbers at least in part via ROS.In addition to GSK3, the activity of C/EBP also appears to be regulated by mTOR, albeit in a different manner.It has recently been shown that the ratio of wild type C/EBP (C/EBPp42) and truncated C/EBPp30, which is generated by alternative translation initiation, is decreased by mTOR, resulting in high levels of the smaller p30 C/EBP isoform (Fu et al., 2010) that inhibits trans-activation of C/EBP target genes in a dominant-negative manner (Pabst et al., 2001) and binds to the promoters of a unique set of target genes to suppress their transcription (Wang et al., 2007).

Deregulated PI3K/PKB signaling in malignant hematopoiesis
The above described studies clearly demonstrate that the PI3K/PKB signaling module plays a critical role in regulation of hematopoiesis.Since constitutive activation of PI3K and/or its downstream effectors has been observed in a high percentage of patients with hematological malignancies, it is likely that the development of leukemia may at least in part depend on aberrant regulation of this signaling module.

PI3K
Constitutive activation of class I PI3K isoforms has been observed in a high percentage of patients with acute leukemia (Kubota et al., 2004;Silva et al., 2008;Billottet et al., 2009;Zhao, 2010).In contrast to the expression of p110, and which is only up-regulated in leukemic blasts of some patients, p110expression appears to be consistently up-regulated in cells from patients with either AML or APL (Sujobert et al., 2005;Billottet et al., 2009).Activating mutations in p110, have been detected in a wide variety of human solid tumors (Ligresti et al., 2009).The most common mutations in p110 are located in the kinase domain (H1047R) and in the helical domain (E545A) (Lee et al., 2005).The E545A mutation has also been detected in acute, but not further specified, leukemia, albeit in a very low percentage (1/88) (Lee et al., 2005).In a series of 44 pediatric T-ALL patients, activating mutations in the catalytic subunit of PI3K (PIK3CA) have been observed in 2 patients, while in frame insertions/deletions have been detected in the PI3K regulatory subunit PIK3R1 in two other patients (Gutierrez et al., 2009).Transplantation of mice with bone marrow cells ectopically expressing mutated p110 resulted in the development of a leukemia-like disease within 5 weeks after transplantation (Horn et al., 2008), suggesting that mutations in p110 would be sufficient to induce leukemia.However, since mutations in PI3K appear to be very rare, it is unlikely that these mutations would be a major cause of leukemic development.Alternatively, the constitutive activation of PI3K observed in many patients with leukemia could also be caused by either aberrant expression or activation of modulators of PI3K activity, including PTEN and SHIP1.Reduced expression of PTEN has, for example, been observed in different types of leukemia (Xu et al., 2003;Nyakern et al., 2006).Both homozygous and heterozygous deletion of PTEN as well as non-synonymous sequence alterations in exon 7 have been detected in approximately 15% and 25% of T-ALL patients, respectively (Gutierrez et al., 2009).In contrast, analysis of both leukemic cell lines and primary AML blasts indicate that PTEN mutations are rare in AML (Aggerholm et al., 2000;Liu et al., 2000).In addition to mutations in PTEN itself, aberrant PTEN expression may also be caused by mutations in its upstream regulators.Both enhanced casein kinase 2 (CK2) expression/activity and enhanced ROS levels appear, for example, to correlate with decreased PTEN phosphatase activity in T-ALL cells (Silva et al., 2008).Both CK2 inhibitors and ROS scavengers were sufficient to restore PTEN activity and impaired PI3K/PKB signaling in those T-ALL cells, demonstrating that aberrant CK2 and ROS levels may affect PI3K signaling in leukemia (Silva et al., 2008).Another important, negative regulator of PI3K activity that has been demonstrated to play a critical role in hematopoiesis is SHIP1.Analysis of primary T-ALL cells revealed that full length SHIP1 expression is often low or undetectable.However, when using an antibody against the C terminal domain of SHIP1, low molecular weight proteins can frequently be observed.These low molecular weight proteins are thought to be the result of mutation induced alternative splicing (Lo et al., 2009).In addition, in leukemic cells from an AML patient, a mutation in the phosphatase domain of SHIP1 has also been detected which results in reduced catalytic activity and enhanced PKB phosphorylation (Luo et al., 2003).For an overview of all known mutations affecting PI3K/PKB signaling, see table 1.

PKB
Constitutive activation of PKB has been demonstrated in a significant fraction of AML patients (Min et al., 2003;Xu et al., 2003;Zhao et al., 2004;Grandage et al., 2005;Gallay et al., 2009).Until recently, no PKB mutations were found in patients with leukemia.However, an activating mutation in the pleckstrin homology domain of PKB (E17K) has recently been detected in solid tumors (Carpten et al., 2007).Transplantation of mice with bone marrow cells ectopically expressing this E17K mutation was sufficient to induce leukemia, ten weeks after transplantation (Carpten et al., 2007).Although this particular mutation has been observed in different types of cancer, it appears to be rare in leukemic patients.Thus far, this mutation has only been detected in one pediatric T-ALL patient (Gutierrez et al., 2009).To date, no other mutations in PKB have been described.Table 1.Mutations in the PI3K/PKB pathway.

Activating mutations upstream of PI3K/PKB signaling pathway
The PI3K/PKB signaling module is an important mediator of cytokine signals.In hematological malignancies, mutations in cytokine receptors have been described to affect PI3K signaling.Constitutive activation of FMS-like tyrosine kinase 3 (FLT3), by internal tandem duplication (Flt3-ITD) (Brandts et al., 2005) and mutation in c-Kit (Ning et al., 2001) have, for example, been demonstrated to induce PKB activity.This induction of PKB activity appears to be essential for the survival and proliferation of cells expressing FLT3-ITD (Brandts et al., 2005) or mutated c-Kit (Hashimoto et al., 2003;Cammenga et al., 2005;Horn et al., 2008).In addition to these tyrosine kinase receptors, the activity of the PI3K/PKB pathway can also be enhanced by several fusion proteins, including Bcr-Abl, which can be detected in virtually all patients with CML (Ben- Neriah et al., 1986) and in patients with ALL (Clark et al., 1988).It has been demonstrated that the PI3K/PKB signal transduction pathway plays an important role in Bcr-abl mediated leukemic transformation (Varticovski et al., 1991;Skorski et al., 1997;Hirano et al., 2009).Other potential regulators of PI3K often mutated in leukemia include Ras (Rodriguez-Viciana et al., 1994; reviewed by Schubbert et al., 2007;Gutierrez et al., 2009) Evi1 (Yoshimi et al., 2011) and PP2A.In AML patients, decreased PP2A activity has, for example, been reported to correlate with enhanced levels of PKB phosphorylation on Thr308 (Gallay et al., 2009).In addition, restoration of PP2A activity also resulted in a reduction of PKB phosphorylation (Cristobal et al., 2011).

Prognosis of acute leukemia with activated PI3K/PKB signaling
As described above, the PI3K/PKB signaling module appears to be aberrantly regulated in a large fraction of patients with leukemia.Recent evidence suggests that the level of PI3K/PKB activation in leukemi c b l a s t s c o u l d b e u s e d t o predict the survival rate of patients.Comparison of pediatric T-ALL patients with either no mutations in PTEN, monoallelic mutations or bi-allelic mutations revealed that the survival rate of patients positively correlates with the level of PTEN (Jotta et al., 2010).Similar observations were made in a different cohort of pediatric T-ALL patients, in which PTEN deletions correlated with early treatment failure in T-ALL (Gutierrez et al., 2009).These studies suggest that constitutive activation of PI3K and its downstream effectors reduces the survival rate of ALL patients.
To determine whether the level of mTOR activity similarly correlates with reduced survival of ALL patients, mice were transplanted with blasts from pediatric de novo B cell progenitor ALL patients.In those experiments, a rapid induction of leukemia correlated with enhanced mTOR activity in the leukemic blasts (Meyer et al., 2011).In addition to ALL, constitutive activation of PI3K, as measured by enhanced FoxO3 expression or phosphorylation, is also considered to be an independent adverse prognostic factor in AML patients (Santamaria et al., 2009;Kornblau et al., 2010).In addition, a reduced survival rate has also been observed in AML patients displaying enhanced levels of phosphorylated, and therefore inactive, PTEN (Cheong et al., 2003) and phosphorylated PKB on Serine 473 (Kornblau et al., 2006) and Threonine 308 (Gallay et al., 2009).In contrast, Tamburini et al. suggest that PI3K activity, as was determined by analysis of the level of phosphorylation of PKB on Ser 473 , positively correlates with the survival of AML patients (Tamburini et al., 2007).Although the short-term survival rate (within 12 months) appeared to be slightly lower in the group displaying high PKB phosphorylation compared to the group with low levels of phosphorylated PKB, both the long-term survival and relapse free survival were significantly enhanced (Tamburini et al., 2007).Except for this last study, all other studies suggest that enhanced PI3K/PKB activity correlates with reduced survival rate in both ALL and AML patients.The molecular mechanisms underlying this reduced prognosis are, thus far, incompletely understood.However, it has been demonstrated that AML blasts displaying enhanced PI3K/PKB activation exhibit a reduced apoptotic response (Rosen et al., 2010) which might be due to positive regulation of the anti-apoptotic NF-kB pathway and negative regulation of the P53 pathway (Grandage et al., 2005).
In addition, since PI3K has been demonstrated to induce expression of the multidrug resistance-associated protein 1 (MRP1), a member of the ATP-binding cassette (ABC) membrane transporters that functions as a drug efflux pump (Tazzari, Cappellini et al. 2007), it could also be hypothesized that constitutive activation of this signaling module results in drug-resistance.The observation that high levels of MRP1 correlates with enhanced drug resistance of AML cells and poor prognosis supports this hypothesis (Legrand et al., 1999;Mahadevan & List, 2004).

PI3K/PKB signaling as therapeutic target in acute leukemia 3.3.1 PI3K inhibitors
Since aberrant regulation of PI3K and its downstream effectors has frequently been observed in leukemic cells and are known to play a critical role in normal hematopoiesis, these molecules are considered to be promising targets for therapy (Table 2).Wortmannin and LY294002 are two well characterized inhibitors of PI3K activity that prevent ATP to bind to and activate PI3K by association with its catalytic subunit (Vlahos et al., 1994;Wymann et al., 1996).Although pre-clinical experiments indicate that both LY294002 and Wortmannin are potent inhibitors of PI3K activity, induce apoptosis in leukemic cells (Xu et al., 2003;Zhao et al., 2004) and rescue drug sensitivity (Neri et al., 2003), it has been demonstrated that both inhibitors exhibit little specificity within the PI3K family and can also inhibit other kinases, including CK2 and smMLCK, respectively (Davies et al., 2000;Gharbi et al., 2007).Since both inhibitors are also insoluble in an aqueous solution (Garlich et al., 2008;Zask et al., 2008) and are detrimental for normal cells (Gunther et al., 1989;Buitenhuis et al., 2008), different PI3K inhibitors are currently developed.Recently, while screening for inhibitors of Cyclin D expression, a novel inhibitor of PI3K activity (S14161) has been discovered that appears to be able to delay tumor growth in mice transplanted with human leukemic cell lines (Mao et al., 2011).In addition, novel inhibitors have been developed that efficiently block the activity of individual p110 isoforms.The p110δ-selective inhibitor IC87114, for example, significantly reduced proliferation and survival of AML blasts (Sujobert et al., 2005) and APL cells (Billottet et al., 2009) without affecting the proliferation of normal hematopoietic progenitors (Sujobert et al., 2005).Similar results were obtained in APL cells treated with an inhibitor directed against p110TGX-115) (Billottet et al., 2009).

PKB inhibitors
In addition to PI3K inhibitors, research has also focused on the development of pharmacological compounds that inhibit its downstream effector PKB.Perifosine, a synthetic alkylphosphocholine with oral bioavailability inhibits PKB phosphorylation by competitive interaction with its PH doma1in (Kondapaka et al., 2003) and promotes degradation of PKB, mTOR, Raptor, Rictor, p70S6K and 4E-BP1 (Fu et al., 2009).In vitro experiments with multidrug-resistant human T-ALL cells and primary AML cells revealed that treatment with Perisofine is sufficient to induce apoptosis (Chiarini et al., 2008;Papa et al., 2008).Moreover, Perifosine reduced the clonogenic activity of AML blasts, but not normal CD34 + hematopoietic progenitor cells (Papa et al., 2008).The efficacy of Perifosine in treatment of different types of leukemia is currently examined in several phase II clinical www.intechopen.com The PI3K/PKB Signaling Module in Normal and Malignant Hematopoiesis 53 trials (NCT00391560, NCT00873457).Phosphatidylinositol ether lipid analogues (PIA) inhibit PKB activity in a similar manner compared to Perifosine.Treatment of HL60 cells with PIA resulted in inhibition of proliferation and sensitization to chemotherapeutic agents in concentrations which did not affect proliferation of normal hematopoietic progenitors (Tabellini et al., 2004).Another specific PKB inhibitor (AKT-I-1/2 inhibitor) (Bain et al., 2007), has been demonstrated to efficiently reduce colony formation in high-risk AML samples (Gallay et al., 2009).The PKB inhibitor Triciribine (API-2), a purine analog that has initially been identified as an inhibitor of DNA synthesis, inhibits PKB phosphorylation by interacting with the PH domain of PKB, thus preventing PKB membrane localization and phosphorylation (Berndt et al., 2010).Experiments in T-ALL cell lines revealed that API-2 induces cell cycle arrest and caspase-dependent apoptosis (Evangelisti et al., 2011a).The safety of this inhibitor is currently under investigation in a phase I clinical trial in patients with advanced hematologic malignancies (NCT00363454).

mTOR inhibitors
Rapamycin and its analogues RAD001 (everolimus), CCI-779 (temsirolimus) and AP23573 (deforolimus) inhibit the mTORC1 complex by association with FKBP-12 which prohibits association of Raptor with mTOR.(Choi et al., 1996;Oshiro et al., 2004).The efficacy of these compounds as therapeutic drugs has been examined in various preclinical and clinical studies for a wide range of malignancies (reviewed by Yuan et al., 2009;reviewed by Chapuis et al., 2010a).The anti-tumor properties of Rapamycin have also been examined in both AML derived cell lines and primary AML blasts, revealing a strong anti-tumor effect of this agent in short-term cultures (Recher et al., 2005).Furthermore, Rapamycin and its analog CCI-779 showed promising effects in preclinical models of T-ALL (Teachey et al., 2008;Meyer et al., 2011) and pre-B ALL (Teachey et al., 2006), respectively.Clinical trials initiated to examine the efficacy of Rapamycin (Recher et al., 2005) and its analog AP23573 in hematological malignancies only resulted in a partial response (Rizzieri et al., 2008).The limited therapeutic effects of Rapamycin and AP23573 may be explained by the induction of PKB activity in AML blasts treated with these compounds (Easton & Houghton, 2006;Tamburini et al., 2008;Yap et al., 2008).Furthermore, experiments with PTEN deficient mice revealed that, due to failure to eliminate the leukemic stem cell population, withdrawal of rapamycin results in a rapid re-induction of leukemia and death in the majority of mice (Guo et al., 2011).This suggests that rapamycin primarily has cytostatic, but not cytotoxic, effects on hematopoietic stem cells.To circumvent the observed up-regulation of PKB phosphorylation by Rapamycin and its analogs, ATP-competitive mTOR inhibitors have been generated that inhibit both the activity of mTORC1 and mTORC2 (Garcia-Martinez et al., 2009;Bhagwat & Crew, 2010;Janes et al., 2010).Treatment of mice transplanted with primary ALL blasts or pre-leukemic thymocytes over-expressing PKB with the mTORC 1/2 inhibitor PP242, but not Rapamycin, significantly reduced the development of leukemia (Hsieh et al., 2010;Janes et al., 2010).
Importantly, PP242 appears to induce less adverse effects on proliferation and function of normal lymphocytes in comparison to Rapamycin (Janes et al., 2010;Evangelisti et al., 2011b).In addition to PP-242, another mTORC1/2 inhibitor, OSI-027, has recently been described.(Evangelisti et al., 2011).It has been demonstrated that this inhibitor exhibits antileukemic effects in both Ph+ ALL and CML cells (Carayol et al., 2010).Furthermore, proliferation experiments indicate that, in comparison to Rapamycin, OSI-027 is a more efficient suppressor of proliferation of AML cell lines (Altman et al., 2011).

Dual inhibition of the PI3K/PKB pathway
In addition to the recently developed mTORC1/2 inhibitors, dual specificity inhibitors have been generated to further optimize inhibition of the PI3K signaling module.PI-103, a synthetic small molecule of the pyridofuropyrimidine class is, for example, a potent inhibitor for both class I PI3K isoforms and mTORC1 (Raynaud et al., 2007).PI-103 has been demonstrated to reduce proliferation and survival of cells from T-ALL (Chiarini et al., 2009) and AML patients (Kojima et al., 2008;Park et al., 2008) and appears to exhibit a stronger anti-leukemic activity compared to both Rapamycin (Chiarini et al., 2009) and the combination of RAD001 and IC87114 (Park et al., 2008).Importantly, although PI-103 reduces proliferation of normal hematopoietic progenitors, survival is not affected (Park et al., 2008).Recently, NVP-BEZ235, another dual PI3K/mTOR inhibitor has been identified.This orally bioavailable imidazoquinoline derivative, has been demonstrated to inhibit the activity of both PI3K and mTOR by binding to their ATP-binding pocket (Maira et al., 2008).
In both primary T-ALL (Chiarini et al., 2010) and AML cells (Chapuis, Tamburini et al. 2010b) as well as leukemic cell lines, NVP-BEZ235 significantly reduced proliferation and survival (Chapuis et al., 2010b;Chiarini et al., 2010).Furthermore, this compound did not affect the clonogenic capacity of normal hematopoietic progenitors (Chapuis et al., 2010b).A dual PI3K/PDK1 inhibitor called BAG956 has also recently been described to inhibit proliferation of BCR-ABL and FLT3-ITD expressing cells.However, in contrast to RAD001 which efficiently reduced the tumor load in mice transplanted with BCR-ABL expressing cells, treatment with BAG956 alone was not sufficient to reduce the tumor load (Weisberg et al., 2008).In addition to these dual inhibitors, KP372-1, a multiple kinase inhibitor capable of inhibiting PKB, PDK1, and FLT3 has been described (Zeng et al., 2006).It has been demonstrated that KP372-1 can induce apoptosis in primary AML cells and leukemic cell lines, as was visualized by mitochondrial depolarization and phosphatidylserine externalization (Zeng et al., 2006).Although the survival of normal hematopoietic progenitors was not impaired by this compound, their clonogenic capacity was, albeit with a low efficiency (Zeng et al., 2006).
In addition to the above described dual inhibitors, the efficacy of combination therapy utilizing multiple inhibitors, which are directed against different intermediates of the PI3K signaling module, is also under investigation.To abrogate the RAD001 mediated upregulation of PKB phosphorylation, the p110 inhibitor IC87114 has, for example, been added to leukemic cells simultaneously with RAD001.Combined inhibition of mTOR and p110 not only resulted in a block in PKB phosphorylation in primary AML blasts, but a synergistic reduction in proliferation could also be observed (Tamburini et al., 2008).
Similarly, combining the PI3K/PDK1 inhibitor BAG956 with RAD001 also resulted in a synergistic reduction in tumor volume in a mouse model transplanted with BCR-ABL expressing cells (Weisberg et al., 2008).Recently, a phase I trial focusing on development of a combination regimen including both perifosine and UCN-01 (NCT00301938), a PDK1 inhibitor which is known to induce apoptosis in AML cells in vitro (Hahn et al., 2005), has been initiated.

Combination of PI3K/PKB pathway inhibitors with other pathway inhibitors
Leukemogenesis involves aberrant regulation of various signal transduction pathways, including, but not limited to, the PI3K signaling module.Simultaneous targeting of multiple  aberrantly regulated signal transduction pathways is considered to be a promising therapeutic strategy (Table 3).Proteosome inhibitors are considered to be a new class of therapeutic agents.However, treatment of both pediatric and adult B-ALL patients with such an inhibitor (Bortezomib) alone was not sufficient to induce a robust anti-tumor response (Cortes et al., 2004;Horton et al., 2007).Experiments in leukemic cell lines and primary cells from B-ALL patients revealed that while MG132, a proteosome inhibitor, and RAD001 alone only modestly reduce cell viability, combined inhibition of proteosomes and mTOR significantly enhanced cell death (Saunders et al., 2011), suggesting a synergistic effect of both inhibitors.In addition to proteosome inhibitors, HDAC inhibitors have also emerged as a promising class of anti-tumor agents (reviewed by Minucci & Pelicci, 2006).Although the HDAC inhibitor MS-275 appears to induce growth arrest, apoptosis and differentiation of leukemic cell lines, in mouse models only a partial reduction in tumor volume could be observed (Nishioka et al., 2008).Combined administration of MS-275 and RAD001, however, potentiated the effect of both inhibitors individually both in vitro and in vivo (Nishioka et al., 2008).Synergistic effects on proliferation and survival of leukemic cell lines have also been observed after coadministration of HDAC inhibitors and the PKB inhibitor Perisofine (Rahmani et al., 2005).Additionally, the efficacy of specific inhibitors targeting constitutively activated tyrosine kinases in leukemia, including inhibitors of Flt3, Abl, and c-Kit, has been investigated in preclinical and clinical models.Although anti-leukemia effects were observed in vivo and in vitro, combined inhibition of tyrosine kinases and the PI3K/PKB pathway resulted in a synergistically enhanced anti-leukemia effect in ALL (Kharas et al., 2008;Weisberg et al., 2008) and AML (Weisberg et al., 2008) compared to the individual inhibitors.Phase I/II clinical trials have already been initiated to investigate the synergistic effects of combined inhibition of PI3K/PKB and Flt3 (NCT00819546) or c-Kit (NCT00762632).

Combination of PI3K/PKB pathway inhibitors with chemotherapeutical agents
Despite the effectiveness of chemotherapy in a subset of patients, incomplete remission and the development of a refractory disease have been observed in many patients with acute leukemia (Thomas, 2009;Burnett et al., 2011).To optimize treatment of those patients, chemotherapy could potentially be combined with leukemia-specific inhibitors or chemosensitizing drugs (Table 3).Co-administration of mTOR inhibitors with different types of chemotherapeutic drugs, including Etoposide, Ara-C, Cytarabine and Dexamethason has, for example, been demonstrated to induce synergistic anti-leukemia effects in cells from AML patients (Xu et al., 2003;Xu et al., 2005) and ALL patients (Avellino et al., 2005;Teachey et al., 2008;Bonapace et al., 2010;Gu et al., 2010;Saunders et al., 2011).Several phase I/II clinical trials have been initiated to investigate and optimize the synergistic effect of mTOR inhibitors and chemotherapeutic drugs in patients with acute leukemia (NCT00544999, NCT01184898, NCT00780104, NCT01162551 and NCT00776373).In addition, co-administration of chemotherapeutic agents with IC87114 (Billottet et al., 2006), UCN-01 (Sampath et al., 2006) or Triciribine (Evangelisti et al., 2011a) showed similar synergistic effects in AML cells.Strong synergistic, cytotoxic, activity was also observed in T-ALL cells when combining the dual specificity inhibitors PI-103 and NVP-BEZ235 with chemotherapy (Chiarini et al., 2009;Chiarini et al., 2010).

Conclusion
During the last two decades, it has become clear that intracellular signal transduction pathways play an important role in both normal and malignant hematopoiesis.One such module implicated in playing a critical role in regulation of various hematopoietic processes includes PI3K and PKB.Aberrant regulation of these molecules appears to be sufficient to induce hematological malignancies.As discussed in this chapter, constitutive activation of this signaling module has been observed in a large group of acute leukemia's.Although activating mutations in PI3K and PKB have been detected in cells from patients with leukemia, these mutations appear to be very rare.In patients, mutations have also been observed in PTEN and SHIP1 resulting in activation of PI3K and its downstream effectors.These mutations, however, cannot account for the large incidence of constitutive activation of PI3K in patients with leukemia.Alternatively, constitutive activation of PI3K and PKB can also be induced by mutations in, for example, tyrosine kinase receptors and by translocation induced formation of fusion proteins.Since PI3K is frequently activated in leukemia and activation of this molecule is thought to correlate with poor prognosis and drug resistance, it is considered to be a promising target for therapy.A high number of pharmacological inhibitors directed against both individual and multiple components of this pathway has already been developed in order to improve therapy.Especially the dual specificity inhibitors seem to possess promising anti-leukemic activities.In addition, research currently focuses on combining inhibitors of the PI3K signaling module with either inhibitors directed against other signal transduction molecules or classic chemotherapy.Mouse models and in vitro experiments indicate that both strategies could be used to improve current therapeutic regimes in specific patient groups.To confirm the pre-clinical data and to examine the safety and efficacy of the individual inhibitors and combination regimes in patients with leukemia, several phase I and II clinical trials have already been initiated.