Leukemia (2010) 24, 1686–1699 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 www.nature.com/leu REVIEW

Perspectives on inhibiting mTOR as a future treatment strategy for hematological malignancies

N Chapuis1,2,3, J Tamburini1,2,4, AS Green1,2, L Willems1,2,4, V Bardet1,2,3, S Park1,2,4, C Lacombe1,2,3, P Mayeux1,2 and D Bouscary1,2,4

1De´partement d’Immunologie-He´matologie, Institut Cochin, Universite´ Paris Descartes, CNRS, UMR8104, Paris, France; 2INSERM, U1016, Paris, France; 3Service d’He´matologie Biologique, Hoˆpital Cochin, AP-HP, Paris, France and 4Service de Me´decine Interne-UF d’He´matologie, Hoˆpital Cochin, Paris, France

Mammalian target of rapamycin (mTOR) is a protein kinase Structure of mTOR implicated in the regulation of various cellular processes, including those required for tumor development, such as the mTOR is a serine/threonine kinase that belongs to the phospho- initiation of mRNA translation, cell-cycle progression and cellular proliferation. In a wide range of hematological malig- inositide 3-kinase (PI3K)-related kinase family and is ubiqui- nancies, the mTORC1 signaling pathway has been found to be tously expressed in mammalian cells. mTOR was first described deregulated and has been designed as a major target for tumor in yeast as the cellular target of rapamycin, an immunosuppres- therapy. Given that pre-clinical studies have clearly established sive agent first identified as a product of the bacterium the therapeutic value of mTORC1 inhibition, numerous clinical Streptomyces hygroscopicus.1 The mTOR protein comprises trials of rapamycin and its derivates (rapalogs) are ongoing a C-terminal catalytic domain, an FRB domain (FKBP-12- for treatment of these diseases. At this time, although disease stabilization and tumor regression have been observed, rapamycin binding domain) that facilitates the binding of objective responses in some tumor types have been modest. FKBP-12(FK506 binding protein 12)/rapamycin complexes, a Nevertheless, some of the mechanisms underlying cancer-cell C-terminal negative regulatory domain, 20 or more N-terminal resistance to rapamycin have now been described, thereby HEAT repeats (Huntingtin, EF3, A subunit of PP2A and TOR) that leading to the development of new strategy to efficiently target enable protein–protein interactions and FRAP-ATM-TRRAP mTOR signaling in these diseases. In this review, we discuss (FAT) domains that modulate catalytic activity.2 It is now known the rationale for using mTOR inhibitors as novel therapies for a variety of hematological, malignancies with a focus on that mTOR resides in at least two distinctive multi-protein promising new perspectives for these approaches. complexes, mTORC1 and mTORC2, which are distinguished by Leukemia (2010) 24, 1686–1699; doi:10.1038/leu.2010.170; their partner proteins, their substrate specificities and their published online 12 August 2010 differential sensitivity to rapamycin. Keywords: mTORC1; mTORC2; PI3K/Akt; hematological malignancies; rapalogs; TORKinhib

mTORC1 Introduction mTORC1 is usually defined as a complex of mTOR and raptor (regulatory-associated protein of mTOR). However, mTORC1 The mammalian target of rapamycin (mTOR) is a key regulator also contains mLST8 (also known as GbL), the proline-rich of various cellular processes needed for growth, cell-cycle 3 Akt/PKB substrate 40kDa (PRAS40) and the recently identified progression and cell metabolism. The mTOR pathway functions dishevelled, egl-10, pleckstrin (DEP)-domain-containing mTOR- as a sensor to ensure an appropriate nutritional state to support 4 interacting protein (deptor). The mTORC1 complex is generally cell development. The blockade of the mTOR signaling can thus inhibited by rapamycin (Sirolimus) and its analogs (CCI-779, prevent cells from responding to extracellular signals transduced RAD001 and AP23573), which are referred to as rapalogs. In from growth factor receptors under conditions of energy and/or most cell types, rapalogs specifically induce the allosteric nutrient deficiency. Conversely, mTOR activation promotes cell repression of mTORC1 activity through their association with growth and proliferation under conditions of energy and nutrient 5 FKBP-12. These FKBP-12/rapamycin complexes bind mTOR in a repletion, through an increase in ribosomal biogenesis and 6 unique region known as the FKBP-rapamycin-binding domain, protein synthesis. As mTOR activity is frequently observed to be 7,8 resulting in the dissociation of mTOR from raptor. The FKBP- deregulated in cancer, including hematological malignancies, 12/rapamycin complexes thus fuction as allosteric inhibitors of this kinase represents a highly attractive target for novel cancer mTORC1 without directly affecting mTOR catalytic activity. therapies. In this review, we discuss the current model for the The exact functions of most mTOR-interacting proteins within mTOR signaling pathway and then focus on the mechanisms the mTORC1 complex remain unclear. Nevertheless, the underlying its deregulation in hematological malignancies. activity of mTORC1 is dependent upon raptor, which functions Finally, we explore the value of targeting mTOR as a future 7,9 as a scaffold for recruiting mTORC1 substrates. The mLST8 anti-cancer therapy. protein lacks kinase activity, but is also required for proper mTOR activity.10 PRAS40 is a negative regulator of mTORC1 Correspondence: Professor D Bouscary, De´partement d’Immunology- and blocks the interaction between mTOR and its substrates. ´ ´ Hematology, Institut Cochin, Universite Paris Descartes, 22 rue Upon phosphorylation by Akt, PRAS40 dissociates from Me´chain, Paris 75014, France. E-mail: [email protected] or [email protected] mTORC1 and binds 14.3.3 proteins, which release mTORC1 11 Received 17 April 2010; revised 7 June 2010; accepted 29 June 2010; activity. Recently a new protein, deptor, has been identified as published online 12 August 2010 an mTOR-interacting protein4 and has been found to negatively Perspectives on inhibiting mTOR N Chapuis et al 1687 regulate mTORC1. Accordingly, the loss of deptor expression (Ras homolog enriched in brain). The active GTP-bound form leads to the activation of mTORC1 signaling.4 of Rheb directly interacts with mTORC1 and stimulates its activity. Accordingly, through its GTPase-activating protein activity TSC1/2 maintains the binding of Rheb to Guanosine mTORC2 diphosphate and suppresses mTORC1 activity (Figure 1). mTORC2 comprises mLST8 and deptor, which are also found Conversely, the activation of Akt downstream of PI3K and in mTORC1, but also specifically contain mSIN1, rictor of the extra-cellular regulated kinase 1/2 (ERK1/2) leads to (rapamycin insensitive companion of mTOR) and protor-1. the inhibition of TSC2 by phosphorylation at its S939/S1462 and Moreover, in contrast to mTORC1, mTORC2 is usually S540/S664 residues, respectively, which leads, therefore, to rapamycin-insensitive,12,13 although some evidence has sug- mTORC1 activation.17–19 gested that prolonged treatment with rapamycin may inhibit its activity towards Akt, depending on the cell type. The induction by rapamycin of an imbalance in the mTORC2 constitution Adenosine mono phosphate-activated protein has been suggested to explain this effect.14,15 Finally, deptor kinase (AMPK) also negatively regulates mTOR activity in mTORC2.4 Energy metabolism has been found to regulate mTOR through the AMPK. AMPK, which is a sensor for cellular energy, responds to the cellular AMP/ATP (adenosine triphosphate) ratio and is Upstream mechanisms leading to mTORC1 activation activated by metabolic stresses that inhibit ATP production (for example, hypoxia and glucose deprivation) or that stimulate ATP Growth factor receptors consumption. Under energy stress conditions, AMPK activates The mTORC1 pathway can be activated either by extracellular TSC2 by phopshorylation on T1227 and S1345,whichleads, signals transduced from growth factor receptors or by modifica- therefore, to the repression of mTORC1 activity.20 tions of the metabolic status of the cell.16 Downstream of growth factor receptors, extracellular signals converge at the tuberous sclerosis complex (TSC1/TSC2/Rheb) axis. TSC1/2 comprises The amino acid pathway hamartin (TSC1) and tuberin (TSC2) and exhibits a GTPase- From a deterministic point of view, it is easy to understand that activating protein function toward the Ras-related GTPase Rheb the rate of protein synthesis is directly dependent upon amino

Amino Energy Growth factors ? acids (ATP)

TKR PIP2 PIP3

308 P IRS T P S473 PI3K PDK1 P Akt AMPK P P P TSC1 TSC2 ERK1/2 Feedback PRAS40 Protor inhibition mLST8 mSIN1 mTOR Rheb GTP mTOR Deptor mLST8 mTORC1 Raptor Deptor Rictor mTORC2

T389 65 37/46 S T P RhoA P P P SGK P P70S6K 70 4E-BP1 T PKCα P Cytoskelatal Protein eIF4E eIF4G P organization synthesis NDRG1 P Paxillin 4E-BP1 Cap-dependent translation eIF4E Cell survival

Figure 1 The mTOR signaling pathway. Red lines indicate the different mechanisms of mTOR activation. Abbreviations: AMPK, AMP-activated kinase; deptor, DEP-domain-containing mTOR interacting protein; 4E-BP1, eIF4E-binding protein 1; eIF, eukaryotic initiation factors; ERK1/2, extra-cellular regulated kinase 1/2; FKBP12, FK506 binding protein 12; IRS, Insulin receptor substrates; mLST8, mammalian lethal with Sec13 protein 8; mTORC, mammalian target of rapamycin complex; NDRG1, N-Myc downstream regulated gene-1; PDK1, phosphoinositide-dependent kinase 1; PI3K, phosphatidylinositol 3-kinase; PIP2, phosphatidylinositol bisphosphate; PIP3, phosphatidylinositol triphosphate; PRAS40, proline- rich Akt1 substrate 1; protor, protein observed with Rictor-1/Proline rich Akt substrate of 40kDa; P70S6K1, p70 S6 kinase 1; Rheb, Ras homolog enriched in brain; mSin1, stress activated protein kinase interaction protein 1; SGK, serum and glucocorticoid protein kinase; TSC, tuberous sclerosis complex; TKR, tyrosine kinase receptor.

Leukemia Perspectives on inhibiting mTOR N Chapuis et al 1688 acid uptake. Among the amino acids, leucine (L) and arginine in inactive complexes. When phosphorylated by mTORC1, (R) are the principal stimulators of mTORC1 activity and the 4E-BP1 dissociates from eIF4E, allowing the interaction between absence of either of them induces the dephosphorylation of eIF4E and eIF4G to occur that then initiates the formation of the mTORC1 substrates, P70S6K and 4E-BP1. In addition, this eIF4F complex (Figure 1). It is of note that this active complex process is dominant over the inputs resulting from growth factor initiates the translation of oncogenic cap-mRNAs, such as MYC, receptors stimulation. Recent evidence further suggests that HIF-1, CCND1 (cyclin-D1) and Bcl-xL.30 signal transduction through amino acids involves the RAG1 and Recent evidence has also emphasized that mTORC1 is an RAG2 proteins, which are small GTPases of the Ras superfamily. important sensor of autophagy, which is stimulated upon RAGs can bind mTORC1 and thereby induce its translocation mTORC1 inhibition.31 Moreover, mTORC1 has been recently to a Rab7-positive late endosomal compartment that contains linked to the control of lipid biosynthesis, a role that may have its Rheb-GTP activator.21,22 Moreover, mTORC1 activation is important implications in cancer biology. Indeed, mTORC1 dependent on glutamine (Q) availability: Q uptake is dependent positively controls the activity of the transcription factors upon the SLC1A5 transporter and in return, Q efflux allows SREBP1 and PPARg, leading to the expression of proteins L import through the antiporter SCL7A5-SCLCA32. The sub- involved in lipid and cholesterol homeostasis.32–34 sequent increase in intracellular L then stimulates mTORC1 activity through a still undefined mechanism.23 New insights into the role of mTORC2 Many important discoveries have been made during the last few Other inputs towards mTORC1 activity years in the field of mTORC2 signaling, which clearly has non- The Wnt signaling pathway, which has generally been reported overlapping functions with mTORC1. This complex does not as a fundamental regulator of the transcription process, has seem to control protein translation and although the mecha- recently been also linked to the control of protein translation. nisms underlying its activity are mostly unknown, mTORC2 has Indeed, Wnt inactivates glycogen synthase kinase 3, which in not been linked either to the cellular energy/nutrient status. It is return blocks TSC1/2 by the direct phosphorylation of TSC2 and, interesting that, however, mTORC2 has a key role in cell thereby activates mTORC1.24 In addition, the p53 protein survival and proliferation through its contribution to the full negatively regulates mTORC1 activity in an indirect manner by activation of the serine/threonine kinase Akt. Indeed, Sarbassov increasing the transcription of the phosphatase and tensin and colleagues have shown in their study that the mTORC2 homolog deleted on chromosome 10 (PTEN), which limits the complex has PDK2 (phosphoinositide-dependent kinase 2) production of PI3K substrates and thus, represses Akt acti- activity and induces the phosphorylation of Akt on its hydro- vity.25,26. Moreover, in response to a genotoxic insult, p53 may phobic S473 residue (Figure 1).35 The phosphorylation status of also activate AMPK, thereby repressing mTORC1 activity.25 Akt, on T308 by PDK1 and on S473 by mTORC2, may govern its substrate specificity. In particular, Akt phosphorylation at its S473 residue is crucial in the control of the transcriptional activity of Upstream mechanisms leading to mTORC2 activation FOXO1 and FOXO3a.10 The inhibition of mTORC2 activity may thus induce FOXO nuclear translocation and the subsequent Owing to its more recent identification, the signals that promote transcription of pro-apoptotic genes. It is of note that, moreover, mTORC2 activity are thus far poorly characterized. Some data when phosphorylated only at T308, Akt retains partial activity strongly suggest that stimulation by growth factors (including against some targets.10 Recently, Alessi and co-workers have insulin) leads to mTORC2 activation through PI3K as the reported that the hydrophobic motif of the serum and pharmacological inhibition of PI3K reduces mTORC2 kinase glucocorticoid protein kinase 1 (SGK1) at S422 is a substrate activity in vitro.27,28 It is interesting that the mSIN protein, a for mTORC2 activity. Upon mTORC2 inhibition, SGK1 activity component of the mTORC2 complex, harbors a PH domain that is fully abrogated, particularly against its direct substrate N-Myc may promote the translocation of mTORC2 to the cell downstream regulated gene-1 (Figure 1).36 Finally, mTORC2 is membrane in a close association with Akt. This event may thus also implicated in the regulation of cytoskeletal organization, participate in the mTORC2-mediated phosphorylation of Akt through i) the phosphorylation of protein kinase C alpha at S657, but further analysis is needed to more clearly understand the ii) the phosphorylation of paxillin at Y118 and iii) the activation mechanisms leading to mTORC2 activation. of the RhoA and Rac1 proteins (Figure 1).10

The mTOR signaling network Deregulation of mTOR signaling in hematological malignancies The established and emerging roles of mTORC1 The mTORC1 complex positively regulates protein synthesis by Most of the available molecular data on hematological malig- activating the ribosomal protein S6 Kinase (P70S6K) and nancies indicate that mTORC1 activity is frequently deregu- inactivating the eukaryotic initiation factor 4E (eIF4E)-binding lated. Moreover, several mechanisms leading to the constitutive proteins (Figure 1). P70S6K is activated by mTORC1 through its activation of mTORC1 have been identified in hematological phosphorylation at T389 and controls the translation of mRNA diseases.37 The best characterized result from the inappropriate molecules harboring 50-terminal oligopyrimidine tracts that are activation of PI3K/Akt signaling mediated by activated tyrosine involved in the biogenesis of ribosomes.29 4E-BP1 is the main kinase receptor and/or by the genetic mutations and amplifica- negative regulator of the initiation of cap-mRNA translation and tion of key components of this pathway.37 However, PI3K, subsequently tightly controls protein synthesis. The mechanism mTORC1 and mTORC2 activities are closely interconnected. It by which 4E-BP1 controls protein synthesis is, however, has been shown both in transformed and in non-transformed complex. Briefly, unphosphorylated 4E-BP1molecules interact cells that mTORC1 negatively regulates PI3K/Akt signaling with the cap-binding protein eIF4E and prevent the formation of because of the phosphorylation of insulin receptor substrate the 4F translational initiation complex (eIF4F) by sequestering it proteins (IRS) by S6K.38 Indeed, IRS-1 and IRS-2 are adapter

Leukemia Perspectives on inhibiting mTOR N Chapuis et al 1689 proteins that are normally required to activate class IA PI3K after mTORC1 inhibitors for specific tumor types and with the stimulation of receptors (for example, by either insulin or appropriate combination of other anti-cancer drugs. Indeed, insulin-like growth factor (IGF-1)). In normal cells, as well as some mechanisms have been suggested to predict the clinical cells in that constitutive mTORC1 activity results from germline response to mTORC1 inhibitors. Accordingly, Shi and colleagues mutations in the TSC1/2 complex, activated P70S6K phosphory- have found that the constitutive activation of PI3K by a PTEN lates the IRS proteins on a serine residue, thereby inducing their mutation could enhance the cellular sensitivity to the rapamycin proteasomal degradation and a decrease in the PI3K/Akt analog CCI-779.42 Frost and colleagues also reported that a activity.39 This negative feedback towards PI3K contributes heightened Akt activity induced hypersensitivity to CCI-779 in a to a reduction in the mTORC1 activity levels and provides myeloma xenograft model.54 This is probably due to the Akt- an auto-regulatory loop for these interconnected pathways. In mediated prevention of vascular endothelial growth factor (VEGF) contrast to mTORC1, mTORC2 increases Akt activation through internal ribosome entry site activity (a cap-independent mechan- its phosphorylation on S473 35 that is necessary for the full ism of mRNA translation) in myeloma cells during mTOR activation of this kinase40 and thus may indirectly increase inhibition that results in a more complete abrogation of VEGF mTORC1 activity. However, no direct deregulation of mTORC2 translation, and ultimately, angiogenesis.55 In contrast, in cells activity has yet been reported in hematological cancers. with a PI3K-independent mTOR activation mechanism, the positive feedback on PI3K/Akt signaling following mTORC1 inhibition may decrease the sensitivity to rapalogs as single agents PI3K-dependent mechanisms and a dual inhibition of both PI3K and mTORC1 signaling The concomitant activation of PI3K/Akt and mTOR signaling is pathways may represent a better strategy.56 The identification of commonly found in hematological malignancies, and PI3K- these predicting factors in the patient population will be essential dependent mechanisms of mTOR activation have been clearly for the success of therapeutic strategies using mTORC1 inhibitors. described. In multiple myeloma (MM) cell lines, the constitutive activation of PI3K can occur through PTEN-inactivating muta- tions or through IGF-1 autocrine production. In these cases, Therapeutic value of mTORC2 inhibition the resulting activation of mTORC1 is fully blocked by PI3K inhibitors.41,42 In addition, the constitutive activation of The critical function of mTORC2 for cancer progression was mTORC1 is frequently detected in chronic myelogenous recently reported by Guertin and colleagues. They showed that leukemia (CML)43,44 and is dependent upon BCR-Abl-induced mTORC2 function is necessary in transformed human prostate PI3K/Akt signaling.43 Accordingly, the BCR-Abl inhibitor epithelial cells, in which the PI3K pathway is aberrantly imatinib represses the activity of mTOR in CML cells.43,45 activated because of PTEN loss, to form tumors when injected into nude mice, whereas the integrity of the mTORC2 complex is not required for normal prostate epithelial cells.57 The PI3K-independent mechanisms selective requirement for mTORC2 during tumor development, In some models, the constitutive activation of mTORC1 does not therefore, suggests that mTORC2-specific inhibitors may be of seem to rely on PI3K activity. In acute myeloid leukemia (AML), substantial clinical utility. Given its reported PDK2 activity,35 the constitutive activation of PI3K is detected in 50% of primary the mTORC2 complex is likely to be activated in most samples, although mTORC1 activity is found in virtually all hematological malignancies with constitutive Akt activation. specimens. Furthermore, the specific inhibition of PI3K activity Although the oncogenic role of mTORC2 in these diseases by IC87114, a specific inhibitor of the p110d isoform of the class remains unclear, suppression of mTORC2-PDK2 activity could IA PI3K that sustains most of the PI3K activity in primary AML have major effects through the inhibition of the oncogenic cells, decreases Akt phosphorylation,46,47 but does not suppress kinase Akt. Moreover, the blockade of mTORC2, resulting in Akt mTORC1 activity.48 Subsequent work by Dos Santos and co- inhibition, may limit the positive feedback loop towards PI3K workers showed that the Src kinase Lyn, which is constitutively resulting from mTORC1 inhibition. This was shown in a study of phosphorylated in most leukemic cells, controls mTORC1, diffuse large B-cell lymphomas, where mTORC2-specific but not Akt activation.49 Similar results were reported by Kharas disassembly by rictor siRNA abrogates rapamycin-induced Akt and colleagues in cell lines derived from a murine model activation.58 of Ph þ pre-B-ALL, which lacks the genes encoding class IA The appropriate way to inhibit mTORC2 is still being debated. PI3K regulatory isoforms. These cells, which are deficient in Although no specific inhibitor selectively targeting the mTORC2 PI3K signaling, show a strong activation of mTORC1 and are complex has yet been developed, the current evidence suggests particularly sensitive to rapamycin.50 In non-Hodgkin lympho- that long-term treatment with rapalogs may inhibit the assembly ma, PI3K-independent mTORC1 activation has also been of the mTORC2 complex in some cell types, including leukemic reported. Leseux and co-workers showed that in follicular myeloid cells.15 Furthermore, second generation of mTOR non-Hodgkin lymphoma cells, the Src kinase Syk influences inhibitors that directly suppress the mTOR catalytic activity in mTORC1 activity independent of PI3K.51 In Mantle Cell both mTORC1 and mTORC2 complexes have been recently Lymphoma (MCL), Akt and mTORC1 are constitutively acti- developed and represent, therefore, a new promising strategy for vated,52,53 but the evidence suggests that the control of mTOR inhibition in hematological malignancies. Finally, Gupta mTORC1 is not exclusively dependent upon PI3K.53 Overall, and co-workers showed in large diffuse B-cell lymphoma, that most of the PI3K-independent mechanisms controlling the the pan histone deacetylase inhibitor LBH589 blocks mTORC2 constitutive activation of mTORC1 remain unknown. activity by inhibiting the interaction between rictor and mTOR.58 Targeting signaling pathways upstream of mTORC2 could be also considered but further work is needed to Mechanisms of mTORC1 activation: predicting the characterize the upstream signaling pathways responsible for sensitivity to mTORC1 inhibitors mTORC2 activation and to understand the oncogenic role of A fuller understanding of the mechanisms that sustain mTORC1 mTORC2 in the initiation and progression of different hemato- activity is clearly required for the successful clinical use of logical malignancies.

Leukemia Perspectives on inhibiting mTOR N Chapuis et al 1690 Targeting mTORC1 signaling with rapamycin and chemotherapy molecules in vivo in NOD/SCID xenotransplant its derivates models, such as rapamycin or CCI-779 in association with methotrexate69 or vincristine in association with RAD001.70 Allosteric mTORC1 inhibitors of the first generation have been Finally, a major role has been revealed for the Notch signaling widely tested as novel drugs to treat hematological malignancies. pathway in a sub-group of T-ALLs. Notch activity is thought to be Apart from the prototypical inhibitor rapamycin, three additional partly mediated by mTORC1 in this subset of T-ALLs, and the derivates (now referred to as rapalogs), namely RAD001 (ever- association of gamma-secretase inhibitors (Notch inhibitors) with olimus), CCI-779 (temsirolimus) and AP23573 (deforolimus), have rapamycin suppresses T-ALL cell growth in a highly synergistic been developed. Rapalogs undergo only minor chemical changes manner.71 It is interesting that mTORC1 inhibition with rapamycin compared with rapamycin, but these modifications lead to the was also able to inhibit growth and proliferation of T-ALL cells increased solubility and stability of these compounds. On the induced by interleukin-4-dependent bone marrow stroma cells basis of promising pre-clinical studies, rapalogs have been used or secretion.72 However, allosteric mTOR inhibitors may eliminate are currently evaluated in a wide range of cancers including ALL tumor cells not by directly inducing apoptosis, but by hematological malignancies.59,60 The scope of the discussion stimulating autophagy. This is not surprising given that mTOR is below is to summarize the pre-clinical and clinical results a gatekeeper that functions upstream of the autophagy machinery obtained with rapalogs, as single agents or in combination with and was recently supported by results showing that RAD001 conventional chemotherapy, particularly in trials for acute increases beclin 1 expression, enhances the levels of LC3-II relative leukemia (AML and ALL), CML, MM, chronic lymphocytic to LC3-1, and finally leads to the punctuate accumulation of acid leukemia (CLL) and MCL. vacuoles, which is indicative of the later stages of the autophagy process.73 Bonapace and colleagues have also recently reported that the association of rapamycin with dexamethasone can trigger Acute myeloid leukemia autophagy-dependent cell death.74 Many clinical studies are Preliminary studies designated the mTORC1 complex as putative currently ongoing to determine the benefit of combining rapalogs target for novel therapies in AML. Xu and colleagues have in a chemotherapeutic regimen to treat relapse/refractory cases of reported that primary AML samples generally show constitutive ALL in both children and adults (Table 1). phosphorylation of the mTORC1 substrates P70S6K and 4E-BP1.61 Moreover, in a xenotransplant model of human AML cells, these authors further found a cooperative anti-tumor effect Chronic myelogenous leukemia of rapamycin and cytarabine.62 Recher and co-workers have Chronic myelogenous leukemia (CML) is a chronic myelopro- described interesting anti-tumor activity of rapamycin as a liferative disorder driven by the expression of the BCR–Abl monotherapy in AML patients, allowing hematological responses fusion protein that results from a t(9;22) translocation. The in nearly 40% among nine patients with refractory/relapsed development of tyrosine kinase inhibitors that target BCR–Abl, AML.63 Furthermore, Zeng and colleagues have shown that both such as imatinib, has brought about a marked improvement in RAD001 and CCI-779 retain the ability to dissociate the mTORC2 the treatment of CML. However, a subset of such patients can complex after a prolonged exposure of AML cells to these acquire resistance to imatinib, mostly through the acquisition of molecules, which decreases the phosphorylation of the mTORC2 BCR–Abl mutations, and new therapeutic agents are, therefore, substrate Akt on S473.15 These authors thus concluded that required to effectively treat CML. mTORC2 disassembly is involved in the anti-leukemic activity of There is much available evidence to show that the mTORC1 rapalogs in AML. Although they are cell-type dependent, similar signaling pathway is consistently activated in CML cells in a results were obtained in our own laboratory, but only in 2/9 BCR–Abl-dependent manner,43 which highlights mTORC1 as a primary AML samples incubated for 24 h with RAD001, even potentially attractive target for CML therapy. Indeed, rapamycin following dose-escalation experiments (unpublished data). not only enhances the imatinib-mediated suppression of The in vitro data for the effects of rapalogs in AML indicate granulocyte macrophage-colony-forming unit progenitors from that these molecules are mainly cytostatic but do not induce patients with CML,45 but also the imatinib-mediated killing of significant levels of apoptosis even after long-term incubation.56 CML cells in vitro.43 Rapamycin also increases the overall Accordingly, previous clinical studies have revealed the low survival of mice in a murine CML model.75 More recently, clinical benefit of rapalogs either alone64 or in combination with Prabhu and co-workers have reported that BCR–Abl positively high-dose chemotherapies65 in refractory/relapsed cases of controls, through the activation of mTORC1, the formation of AML. However, although the anti-leukemic activity of the the eIF4F translation initiation complex and subsequent cap- rapalogs has produced largely disappointing results in clinical mRNA translation.76 Moreover, other pre-clinical data have practice, in vitro studies are ongoing to identify compounds that revealed that mTOR inhibition may constitute a promising may function synergistically with them in AML, as reported for therapeutic target in CML patients showing resistance to the use of histone deacetylases inhibitors (Table 1).66 imatinib. Indeed, Ly and colleagues reported that rapamycin can overcome this resistance in CML cells harboring a BCR–Abl gene amplification,43 and Sillaber and co-workers have also Acute lymphoid leukemia recently shown that rapamycin inhibits the growth of cells with Early reports suggested that the targeting of mTORC1 with imatinib-resistant mutants of BCRFAbl, including the highly rapalogs could be of therapeutic value in the treatment of acute aggressive T315I variant.77 Finally, mTOR signaling inhibition lymphoblastic leukemia (ALL). Avellino and colleagues reported may induce a decrease of angiogenesis in CML as it has been that rapamycin increases the anthracyclin-induced apoptotic shown that mTORC1 was implicated in the BCR–Abl-dependent response in ALL cells in vitro.67 Teachey and co-workers have expression of VEGF and its transcriptional activator, hypoxia shown that CCI-779 alone reduces cell proliferation and induces inducible factor-1 (HIF-1).78 However, although pre-clinical apoptosis in ALL and inhibits the growth of human ALL cells data strongly support the rationale for the use of rapalogs in the in vivo in NOD/SCID xenotransplant models.68 In addition, treatment of CML, few patients have been treated with these reported evidence suggests that rapalogs synergize with key ALL compounds as the inhibitors in current use to treat this cancer

Leukemia Perspectives on inhibiting mTOR N Chapuis et al 1691 Table 1 Recent clinical trials of rapalogs in the treatment of hematological malignancies

Clinical Trials.gov Disease Phase Investigator location Details Status Number

Groupe Ouest Est d'Etude des Leucémies et Everolimus in association with Aracytine and Daunorubicine in AML treatment in patients AML NCT01074086 I Recruiting Autres Maladies du Sang GOELAMS less than 65 years in relapse more than one year after first complete remission

AML NCT00636922 I Bayside Health Everolimus in Elderly Patients Wi th AML Unfit for Intensive Induction Chemotherapy Recruiting

Institut de Recherches sur les Leucemies et Phase I Study Evaluating the Chemosensitizing Effect of Everolimus Administered With AML NCT00544999 I Recruiting les Maladies du Sang Cytarabine and Daunorubicin in Patients With Acute Myeloid Leukemia in Relapse

Everolimus in Combination with PKC412 in patients with relapsed, refractory or poor AML NCT00819546 I Dana-Farber Cancer Institute Recruiting prognosis AML or MDS

Medical faculty of the Technical University AML NCT00762632 I/II Combination of Nilotinib (AMN 107) and Everolimus in patients with AML Recruiting Munich

AML NCT00775593 II Ospedale Sant' Eugenio Clofarabine and Temsirolimus in older patients with relapsed or refractory AML Recruiting

ALL NCT00968253 I/II UT MD Anderson Cancer Center Everolimus for patients with relapsed or refractory ALL Recruiting

Everolimus in combination With Imatinib (Glivec®/Gleevec™) in patients with CML in CML NCT00093639 I/II Roswell Park Cancer Institute Complete chronic phase who are not in complete cytogenetic response to imatinib-alone

CML NCT00101088 I Duke-NUS Graduate Medical School Temsirolimus in combination with Imatinib Mesylate in CML Recruiting

Treatment of Relapsed/Refractory Chronic Lymphocytic Leukemia/Small Lymphocytic CLL NCT00935792 I/II Mayo Clinic Recruiting Lymphoma With Everolimus (RAD001) and Alemtuzumab

Active, not CLL NCT00290472 II M.D. Anderson Cancer Center A Phase II Study of CCI-779 in B-Cell Lymphoma and Chronic Lymphocytic Leukemia recruiting

A Phase II Study of CCI-779 in Patients With Relapsed, Refractory or Transformed CLL NCT00086840 II M.D. Anderson Cancer Center Complete Chronic Lymphocytic Leukemia

MCL NCT00787969 I/II Mayo Clinic Rituximab, Cladribine, and Temsirolimus in treating patients with newly diagnosed MCL Recruiting

Active, not MCL NCT00117598 III Wyeth Temsirolimus in relapsed refractory Subjects with MCL recruiting

Active, not MCL NCT00109967 II Mayo Clinic Temsirolimus in combination With Rituximab in patients with relapsed or refractory MCL recruiting

MCL NCT00033267 II Mayo Clinic Temsirolimus in previously treated patients with MCL Complete

MCL NCT00516412 II UniversitaetsSpital Zuerich Everolimus for the treatment of patients With relapsed or therapy resistant MCL Recru iting

Everolimus as maintenance therapy for patients Above 60 Years in MCL after first and MCL NCT00727207 II University of Munchen Recruiting second line chemotherapy

Everolimus in Combination with Lenalidomide in the treatment of subjects with relapsed MM NCT00729638 I Massachusetts General Hospital Complete and relapsed/refractory MM

Active, not MM NCT00618345 II Mayo Clinic Everolimus in relapsed/refractory MM recruiting

Active, not MM NCT00483262 I/II Dana-Farber Cancer Institute Combination of temsirolimus and Velcade in relapsed and/or relapsed/refractory MM recruiting

MM NCT00693433 I Jonsson Comprehensive Cancer Center Temsirolimus in Combination With Dexamethasone in MM Recruiting

Arthur G. James Cancer Hospital & Richard Active, not MM NCT00398515 I Lenalidomide and Temsirolimus in Patients with relapsed or refractory MM J. Solove Research Institute recruiting

Arthur G. James Cancer Hospital & Richard MM NCT00079456 II Temsirolimus In patients with relapsed or refractory MM Complete J. Solove Research Institute

Abbreviations: AML, acute myeloid leukemia; ALL, acute lymphoid leukemia; CML, chronic myelogenous leukemia, MCL, mantle cell lymphoma; MM, multiple myeloma; CLL, chronic lymphocytic leukemia. Ongoing clinical trials involving rapalogs were retrieved from www.clinicaltrials.gov in March 2010. Only those studies focused on a specific hematological malignancy are included. are extremely safe and efficient. In a previous study, six patients Multiple myeloma with imatinib-resistant CML were administered rapamycin at The PI3K/Akt and mTOR signaling pathways are frequently 2 mg per day for 14 consecutive days and a major leukocyte activated in MM cells and many cytokines or growth factors, response (GBo10G/l) occurred in two cases (33%).77 Clinical including interleukin-6 or IGF-1, can stimulate the activity of the studies of rapalogs in imatinib-resistant and/or accelerated PI3K/Akt/mTORC1 axis in MM cells. Accordingly, the inhibition phase CML patients are ongoing (Table 1). of IGF-1R signaling with the IGF-1R kinase inhibitor

Leukemia Perspectives on inhibiting mTOR N Chapuis et al 1692 NVP-AEW541 has additive/synergistic effects with the mTORC1 malignant B cells. However, evidences recently showed the inhibitor RAD001 in myeloma cells.79 Heightened Akt activity existence of a proliferating pool of malignant B cells in lymph in MM cells may also sensitize them to the effects of the nodes and bone marrow. As constitutive activation of mTOR mTORC1 inhibitors rapamycin or CCI-779.42,54,55 It has been signaling pathway is frequently detected in primary CLL cells,88 further observed that rapalogs block cyclin D1 expression more mTOR targeted therapy could, therefore, represent a new profoundly in MM cells that show high Akt activity, due to the attractive strategy in CLL by affecting proliferation of the pool inhibition of cyclin D1 mRNA internal ribosome entry site of cycling cells. It is interesting that pre-clinical studies showed activity in ‘high Akt’ myeloma cells.80 that rapamycin induced a cell-cycle arrest of proliferating B-CLL Very recently, a new regulator of mTOR, deptor, has been cells through the reduction of Cyclin D3, cyclin E, cyclin A and identified in MM cells. Deptor is generally expressed at high surviving expression89 without affecting non-malignant B cells. levels in MM cell lines in contrast to other cancers and is also Furthermore, other studies recently reported a pro-apoptotic overexpressed in 28% of human primary MM cells.4 Deptor effect of rapamycin in primary B-CLL cells, thereby emphasizing expression is negatively regulated by mTORC1 and mTORC2 the therapeutic value of mTOR inhibition in this disease.90,91 and this protein in turn inhibits these complexes.80 Moreover, Finally, Zanesi and colleagues showed the ability of rapamycin although it blocks protein translation through the inhibition of to improve survival of CLL transgenic mouse.92 Given these mTORC1, deptor increases PI3K/Akt activity by a S6K-depen- results, clinical trials are ongoing in CLL and results from two dent increase in IRS-1 expression that stimulates IGF-1 signal- phase II clinical trials have been recently published. Decker and ing.81 This dual and opposing regulation of mTORC1 and colleagues tested the effect of RAD001 5 mg per day in patients mTORC2 activities by deptor (that is, maintaining low with advanced B-CLL and reported one partial response and mTORC1, but high mTORC2 levels) may be specifically three patients with stable disease.93 However, this trial was necessary in MM cells given their high demand on protein stopped after treatment of seven patients because of the synthesis that causes a significant level of endoplasmic development of opportunistic infections. In the study of Zent reticulum stress. Low mTORC1 activity, through the limitation and colleagues, 22 patients with refractory CLL were treated of protein synthesis, may thus be viewed as a mechanism that with everolimus 10 mg per day and a partial remission was limits the level of endoplasmic reticulum stress below the observed in 4 patients (18%). Unexpectedly, the authors also threshold at which apoptosis is triggered. observed an increase in absolute lymphocyte count associated The currently available data from pre-clinical models strongly with a decrease in lymphadenopathy in eight patients (36%), indicate that the rapalogs have anti-myeloma properties. Indeed, thereby suggesting the ability of everolimus to mobilize rapamycin induces both cell-cycle arrest and significant malignant cells from nodal masses into the peripheral circula- apoptosis in MM cells.82 Yan and colleagues have shown in a tion.94 Combinations with other therapeutic regimens are, xenograft model that CCI-779 enhances the responsiveness of therefore, currently tested in other clinical trials (Table 1). myeloma cells to dexamethasone in vivo through the inhibition of cap-mRNA translation.83 A synergistic anti-myeloma effect was also observed in vitro using a combination of rapamycin Mantle cell lymphoma and the HSP90 inhibitor 17-AAG.84 A previous study from Raje One of the most promising areas of study in mTOR inhibition and co-workers has provided a rationale for the association of has involved MCL. MCLs are characterized by a t(11;14) rapamycin with immunomodulatory compounds, such as translocation, leading to cyclin D1 overexpression. As cyclin lenalidomide, which may overcome the resistance in myeloma D1 expression is tightly regulated at the translation initiation cell lines to conventional chemotherapeutics.85 Furthermore, level by mTORC1,30 MCL was thought likely to have the rapamycin combined with lenalidomide was able to overcome greatest potential for a positive clinical outcome as a result of the growth advantage conferred on MM cells by interleukin-6, targeting mTOR. However, a number of different studies have IGF-1 or adherence to bone marrow stromal cells.85 Finally, the reported that rapalogs fail to suppress cyclin D1 expression in therapeutic value of mTOR inhibition in MM could be due to MCL cells.95–97 In each of these reports, it was shown that the decrease of pathological angiogenesis. Indeed, Frost and rapalogs block MCL cell proliferation leading to an accumula- collegues clearly showed that CCI-779 inhibits VEGF translation tion of G1-phase cells, while only inducing a weak pro- resulting in a decrease of angiogenesis.55 It is interesting that apoptotic effect. Pre-clinical work has also shown that rapalogs mTOR was reported to be implicated in another mechanism can exert synergistic effects in vitro with classical agents used in contributing to angiogenesis in MM, as conditioned media of MCL therapy, such as rituximab, vincristine and doxorubicin.98 MM cells induced an mTOR-dependent increase in expression On the basis of these previous data, a number of clinical trials of VEGF receptors (VEGFR-1 and VEGFR-3) on human bone of rapalogs have been conducted and it is of some interest that marrow endothelial cells.86 clinical successes have been reported in patients with MCL. Two Taken together, these studies provide significant insight into phase II trials, conducted previously by Witzig and colleagues99 the importance of the mTOR pathway for the cell growth and and Ansell and co-workers,100 tested the effects of 250 mg or survival of myeloma cells and have led to the design and 25 mg of CCI-779, respectively, when administrated intrave- implementation of a number of clinical trials. Recently in this nously every week as a single agent in patients with relapsed regard, 16 patients with relapsed/refractory MM received a MCL. Both studies reported a similar overall response rate weekly i.v. dose of 25 mg of CCI-779 in a phase II trial and an (38 and 41%) indicating that as a single agent, CCI-779 could be overall response rate of 38% was observed.87 Other clinical an effective choice as an MCL therapy. AP23573 was also tested trials of rapalogs in combination with conventional agents are in patients with MCL and in nine patients who received 12.5 mg currently underway (Table 1). of this agent once daily for 5 days every 2 weeks, a partial response was obtained in three (33%) cases.101 More recently, a phase III study conducted by Hess and colleagues has evaluated Chronic lymphocytic leukemia two different dose regimens of CCI-779 in comparison with B-cell CLL is the most common form of leukemia and classical treatments for relapsed and refractory hematological is characterized by the progressive accumulation of small malignancies. The conclusion of this study was that CCI-779 at

Leukemia Perspectives on inhibiting mTOR N Chapuis et al 1693 175 mg weekly for 3 weeks followed by 75 weekly treatments disappointing, particularly in the case of AML. Efforts have thus significantly improved the progression-free survival and overall been made to better elucidate the mechanisms underlying the response rate compared with the control group.102 rapamycin-resistance phenotype. It must be noted however that the biological mechanisms underlying the clinical anti-tumor activity of CCI-779 remain unclear at present, as this compound barely induces apoptosis in mTOR feedback loops MCL cells ex vivo. It has been reported that CCI-779 induces As reported in non-transformed cells, stimulation of the PI3K/Akt autophagy, which was attested by the increased formation of pathway has been extensively described in response to acidic vesicular organelles and processing of LC3 in MCL cells mTORC1 inhibition, in AML,56 MM,81 MCL104 and also in following exposure to this agent.103 Hence, prolonged cell-cycle BCR–Abl and ALL.50 Indeed, by inhibiting the mTOR-dependent arrest or autophagy induced by CCI-779 can trigger cell serine phosphorylation of the IRS1/2 adaptor molecules down- death.97,104 Moreover, rapalogs may block angiogenesis through stream of tyrosine kinase receptors, rapalogs can enhance PI3K/ the inhibition of VEGF production and this may be particularly Akt signaling (Figure 2).38 This clearly decreases the anti-tumor important in highly angiogenic tumors, such as MCL and non- efficacy of rapalogs in vitro by increasing pro-proliferative inputs Hodgkin lymphoma.105 Other ongoing studies are currently that may have clinical relevance, although this is yet to be underway to test the efficacy of temsirolimus in combination shown. Hence, the aforementioned results provide a rationale with rituximab (Table 1). for the concomitant inhibition of mTORC1 and PI3K activity in these diseases (Figure 3a). Accordingly, we and others have shown that the dual PI3K and mTORC1 inhibitor PI-103106 Overcoming rapamycin resistance blocks the overactivation of PI3K/Akt that results from mTORC1 inhibition and induces the death of primary AML cells ex vivo, Rapalogs have shown promise as anti-cancer therapeutics for a which sharply contrasts with the results obtained with RAD001 wide range of hematological malignancies particularly in the or IC87114 (a specific p110d inhibitor) alone.107,108 In B-ALL, case of lymphoid neoplasms. However, the results of clinical Kharas and colleagues have shown that PI-103 is more efficient trials of these agents have been heterogeneous and mostly than rapamycin at suppressing the proliferation of human ALL

Growth factors

TKR (IGF-1R) T308 P S473 IRS P Cell proliferation PI3K Akt and survival (4)

P (7) ERK1/2 TSC1 TSC2

(3)

mTOR Rheb GTP mTOR Rictor

mTORC1 Raptor (1) (8) mTORC2 (5) (2) Rapalogs 389 T S65 T37/46 P P P FKBP12 P70S6K 4E-BP1 T70 P eIF4E eIF4G

4E-BP1 s209 P eIF4G Cap-dependent eIF4E eIF4E translation (6) 7mGTP mRNA

Figure 2 Mechanisms underlying the rapamycin-resistant phenotype. Different mechanisms have been proposed to explain the emergence of rapalog-resistance in hematological malignancies. Rapalogs associate with FKBP12 and (1) dissociate the mTORC1 complex that leads to (2) the inhibition of P70S6K phosphorylation. This results in (3) the suppression of the negative input from mTORC1 toward PI3K/Akt and leads to (4) the overactivation of Akt. Another mechanism that leads to rapalog resistance involves the deregulation of protein translation in certain hematological malignancies, such as AML. In contrast to P70S6K, (5) rapalogs do not suppress 4E-BP1 phosphorylation events, and thus allow active cap-mRNA translation. Cap-mRNA translation is also increased through (6) rapalog-induced eIF4E S209 phosphorylation, which enhances its affinity for the 7mGTP cap structure of mRNA. Rapalogs also induce (7) an increase of ERK/MAPK signaling pathway. Finally, mTORC2 activity is generally reported to be rapamycin-insensitive in most models (8). The red broken lines indicate the inhibitory effects mediated by rapalogs. Red arrows indicate activating effects induced by rapalogs.

Leukemia Perspectives on inhibiting mTOR N Chapuis et al 1694 abGrowth factors Growth factors (1) Anti-IGF-1R TKR TKR IGF-1R (1) (IGF-1R) TKI (IGF-1R) P IRS P IRS

PI3K (2) Dual PI3K (2) PI3K inhibitors inhibitors T308 308 473 T 473 P P S P P S ERK Akt mTORC2 Akt inhibitors mTORC2 mTOR (3) mTOR Rictor Rictor ERK1/2 TSC1 TSC2 TSC1 TSC2 (1)

TORKinhib

mTOR mTOR

mTORC1 Raptor mTORC1 Raptor Rapalogs

FKBP12 389 389 T S65 T37/46 T S65 T37/46 P P P P P P P70S6K 4E-BP1 P70S6K 4E-BP1 T70 T70 P P

Figure 3 Possible new options for the inhibition of mTOR in hematological malignancies. (a) To enhance their anti-tumor activity, rapalogs can be combined with (1) IGF-1/IGF-1R signaling inhibitors, such as anti-IGF-1R antibody or IGF-1R Tyrosine Kinase Inhibitors (TKI); (2) PI3K inhibitors; or (3) ERK/1/2 inhibitors. (b) The complete inhibition of mTOR catalytic activity in both mTORC1 and mTORC2 complexes could have more potent anti-tumor effects. This could be achieved using (1) second generation mTOR inhibitors (‘TORKinhibs’) or (2) dual PI3K/Akt and mTOR inhibitors. Red broken lines indicate the effects induced by these different inhibitors.

leukemia cells treated with imatinib.50 PI-103 has also been enhances its affinity for the 7mGTP cap structure of mRNA and tested in primary samples and in cell lines derived from T-ALL increases protein translation (Figure 2). This process implicates a cells. This compound has been found to reduce leukemic-cell PI3K-dependent, but Akt-independent activation of Mnk-1/2 proliferation, block the cell cycle and induce apoptosis, and kinases as the underlying mechanism.114 We have also observed consistently, show stronger anti-leukemic properties compared a similar PI3K-dependent RAD001-mediated increase in eIF4E with rapamycin.109 Dual PI3K/Akt and mTOR inhibitors have S209 phosphorylation in primary AML cells, but the participation also been tested against MM. McMillin and co-workers recently of this process in the rapamycin-resistance phenotype is still reported the activity of the NVP-BEZ235 compound (Novartis, unclear in AML (unpublished data). As another example, Basel, Switzerland) in vivo in a mouse xenograft model of MM a previous study by Carracedo and colleagues has shown a as single agent and in combination with clinically available similar feedback loop in the ERK/MAPK pathway following molecules (bortezomib, doxorubicin and dexamethasone).110 immunohistochemical analysis of tissue samples from breast Another approach for abrogating the positive feedback from cancer patients treated with RAD001 (Figure 2). The association PI3K/Akt signaling due to mTORC1 inhibition is by the conco- of a MEK1/2 inhibitor (UO126) with RAD001 was found to mitant inhibition of the IGF-1/IGF-1R pathway (Figure 3a). enhance its anti-tumor effects in vitro (Figure 3a).115 Preliminary In AML, we56 and others111,112 have reported that an IGF-1 results from our laboratory suggest that a similar process could autocrine production is responsible for both PI3K constitutive occur in AML (personal unpublished data). activity113 and the overactivation of PI3K/Akt signaling observed after RAD001 treatment.56 We thus hypothesized that simulta- neous blocking IGF-1/IGF-1R and mTORC1 could enhance the Deregulation of the cap-mRNA translation process anti-leukemic activities of each therapy, although experimental Another mechanism leading to rapamycin resistance is the evidence is lacking to support this theory in AML. In MM, deregulation of protein translation. Indeed, we recently reported Baumann and co-workers have reported that a combined that protein synthesis is rapamycin-resistant in primary AML treatment with the IGF-1R tyrosine kinase inhibitor NVP- cells and that rapamycin does not suppress the 4E-BP1 AEW541 (Novartis) and RAD001 is effective and could phosphorylation events that control cap-mRNA dependent represent a new therapy option for this disease.79 translation (Figure 2).48 The rapamycin-resistant phosphoryla- Additional feedback mechanisms involving PI3K upon tion of 4E-BP1, which is absolutely required for the formation of mTORC1 inhibition have been described in a number of cancer the EIF4F translation initiation complex, is mostly controlled cell lines. For example, rapamycin treatment increases eIF4E by the Pim-2 kinase in primary AML cells.48 Consequently, S209 phosphorylation in lung and breast cancer cell lines, which Pim-2 overexpression may allow protein translation in AML

Leukemia Perspectives on inhibiting mTOR N Chapuis et al 1695 cells treated with rapalogs, most notably the expression of Dual ATP-competitive inhibitors of PI3K and mTOR, such as oncogenic proteins, such as c-Myc, cyclin D1 or Bcl-xL. These PI-103 have already been tested in different hematological results also emphasize the rationale for a direct blockade of malignancies (Figure 3d).50,107,109 In addition, several other translation regardless of mTORC1 inhibition as a treatment direct PI3K and mTOR-targeting compounds have been recently strategy for AML. Indeed, we have shown that the specific reported, including NVP-BEZ235 (Novartis). It is interesting that inhibition of the association between eIF4E and eIF4G by we have reported that this compound strongly inhibits 4E-BP1 the 4EGI-1 compound suppresses the formation of the eIF4F phosphorylation and cap-dependent mRNA translation in translation initiation complex that strongly induces the apoptosis primary AML cells,123 which provided a rationale for develop- of leukemic cells, while sparing normal CD34 þ hematopoietic ment of further clinical trials with this molecule for AML. It is of progenitors.48 interest in this regard that McMillin and co-workers tested NVP- BEZ235 in MM cells and observed marked anti-myeloma Lack of mTORC2 inhibition activity with a favorable therapeutic index in both 110 As mentioned above, mTORC2 activity is generally reported to in vitro experiments and in vivo xenograft studies. Baumann be rapamycin-insensitive (Figure 2). However, long-term treat- and colleagues have also tested NVP-BEZ235 in MM cells and ments with rapalogs can dissociate the mTORC2 complex, showed cooperative activity with melphalan, doxorubicin and 124 resulting in a decrease in Akt S473 phosphorylation in some cell bortezomib. Finally, NVP-BEZ235 was recently found to types, including leukemic cells.15 However, the relevance of have a significant efficiency in a PEL (Primary effusion 125 this phenomenon to hematological malignancies biology is lymphoma) xenograft tumor model. unknown at present. Whether dual PI3K/mTOR inhibitors are superior to TORkinhibs as anti-cancer drug still remains unclear. When In summary, mTORC1-inhibition using rapalogs results in the PI3K/Akt and mTOR are activated separately, which is the activation of multiple feedback loops involving oncogenic case for example in AML,48 we can predict a superior outcome kinases, such as PI3K or ERK that may limit the activity of these for dual inhibitors compared with TORkinhibs. However, as molecules. The anti-tumor effects resulting from the concomitant suggested by Janes and colleagues in their ALL BCR–Abl þ cell use of rapalogs combined with other targeted molecules (for model, although both inhibitors have a similar degree of anti- example, Pan PI3K or specific class IA PI3K inhibitors, ERK/MAPK leukemic efficacy, dual PI3K/mTOR inhibitors may cause a inhibitors or IGF-1R inhibitors) or direct inhibition of the greater degree of immune suppression by affecting the proli- translation machinery downstream of mTORC1, therefore, seems feration and function of normal lymphocytes.121 to have much therapeutic promise for hematological malignancies and warrants further testing using in vitro models of these cancers.

New options for mTOR inhibition using ‘TORKinhibs’ Conclusion

A promising approach for overcoming the above mentioned The activity of the oncogenic kinase mTOR is frequently limitations of the rapalogs is the use of ATP competitive detected in hematological malignancies. However, the exact ‘active-site’ mTOR inhibitors (Figure 3b). These second genera- frequencies of mTORC1 and mTORC2 constitutive activation, tion mTOR inhibitors, referred to as ‘TORKinhibs’, specifically as well as their specific roles in cancer initiation, progression block the serine/threonine kinase activity of mTOR in a FKBP- and maintenance, need to be specifically addressed in different 12-independent manner, which is in contrast to rapalogs. neoplasms. The mTORC1-dependent signaling pathway is Recently, different groups have reported the identification and engaged in complex interactions with the PI3K/Akt axis and characterization of those molecules.116–120 It is interesting that the inhibition of mTORC1 by rapalogs can lead to PI3K/Akt TORkinhibs strongly suppress 4E-BP1 phosphorylation and overactivation. This underlies the rationale for dual PI3K and inhibit the phosphorylation of mTORC2 substrates, including mTORC1 inhibition, particularly when mTORC1 activity is not Akt and SGK1, without affecting PI3K activity.117,121 Hence, driven by PI3K/Akt. It has also emerged from in vivo studies that more efficient anti-cancer activities can be obtained both the sensitivity to rapalogs is both tumor- and cell type-specific in vitro and in vivo with these second generation mTOR and that determining the molecular basis for this heterogeneity inhibitors compared with rapalogs through the full suppression represents a major challenge for future studies. In addition to the of both mTORC1 and mTORC2 signaling and probably also by blockade of cell proliferation, rapalogs may also control the strong blockade of protein translation. Accordingly, Janes autophagy, which is a bidirectional process that either promotes and colleagues have previously tested the PP242 compound in cell survival or induces autophagic cell death. An increased leukemic cells expressing the BCR-Abl oncogene and showed understanding of how to further drive cancer cells exposed to that, in contrast to rapamycin, this compound inhibits both rapalogs to autophagic death may assist with the development of mTORC1 and mTORC2 activity and suppresses 4E-BP1 phos- new therapies. Finally, first generation mTOR inhibitors or phorylation. This induces leukemic-cell death and enhances the rapalogs, acting through the allosteric inhibition of mTORC1 anti-leukemic activity of dasatinib in vitro and in vivo, effects of activity, will almost certainly be supplanted by a second which are superior to those obtained with rapamycin.121 generation of these molecule or TORKinhibs, which also target Furthermore, Hsieh and colleagues also recently reported, in the second mTOR-containing complex mTORC2, which now a transgenic mouse model in which constitutively activated has a clearly established role in cancer biology. Overall, forms of Akt1, Akt2 or Akt3 were expressed in immature T cells, targeting mTOR with these new pharmacological tools will the development of spontaneous thymic lymphomas and preserve the anti-proliferative activity of rapalogs, while the therapeutic efficacy of the PP242 compound through the enlarging their anti-cancer spectrum by the blockade of protein blockade of 4EBP-eIF4E hyperactivation.122 However, the translation, ultimately inducing death in the transformed cells. precise role of these second generation mTOR inhibitors in Future clinical trials with these new drugs will almost certainly hematological malignancies remains to be fully determined provide exciting new perspectives in the development of novel in large-scale clinical studies in the near future. treatments for hematological malignancies.

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