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Keywords:

  • mammalian target of rapamycin;
  • phosphatidylinositol 3′ kinase;
  • protein kinase B;
  • phosphatase and tensin homologue tumor suppressor;
  • CCI-779;
  • RAD001;
  • AP23573

Abstract

  1. Top of page
  2. Abstract
  3. The Role of mTOR in Cellular Signaling
  4. Proteins in the Pathway Proximal to mTOR
  5. Proteins in the Pathway Distal to mTOR
  6. mTOR Signaling Pathway Dysregulation, Cell Transformation, and Antineoplastic Effects of mTOR Inhibition
  7. mTOR Inhibitors in Clinical Development in the Hematologic Malignancies
  8. CONCLUSIONS
  9. REFERENCES

The mammalian target of rapamycin (mTOR) is a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signaling pathway, which mediates cell survival and proliferation. mTOR regulates essential signal-transduction pathways, is involved in the coupling of growth stimuli with cell cycle progression, and initiates mRNA translation in response to favorable nutrient environments. mTOR is involved in regulating many aspects of cell growth, including membrane traffic, protein degradation, protein kinase C signaling, ribosome biogenesis, and transcription. Because mTOR activates both the 40S ribosomal protein S6 kinase (p70s6k) and the eukaryotic initiation factor 4E-binding protein 1, its inhibitors cause G1-phase cell cycle arrest. Inhibitors of mTOR also prevent cyclin dependent kinase (CDK) activation, inhibit retinoblastoma protein phosphorylation, and accelerate the turnover of cyclin D1, leading to a deficiency of active CDK4/cyclin D1 complexes, all of which may help cause G1-phase arrest. It is known that the phosphatase and tensin homologue tumor suppressor gene (PTEN) plays a major role in embryonic development, cell migration, and apoptosis. Malignancies with PTEN mutations, which are associated with constitutive activation of the PI3K/Akt pathway, are relatively resistant to apoptosis and may be particularly sensitive to mTOR inhibitors. Rapamycin analogs with relatively favorable pharmaceutical properties, including CCI-779, RAD001, and AP23573, are under investigation in patients with hematologic malignancies. Cancer 2004;100:657–66. © 2004 American Cancer Society.

To control carefully the many processes involved in growth, eukaryotic cells have evolved an array of interlinked regulatory mechanisms to modulate protein synthesis and degradation in response to a variety of stimuli. Regulation of cellular protein synthesis plays a critical role in development, differentiation, cell cycle progression, growth, and apoptosis.1 The mammalian target of rapamycin (mTOR) is involved in regulating many aspects of cell growth as well as cell cycle progression, membrane trafficking, protein degradation, and both protein kinase C signaling and transcription (Fig. 1).2

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Figure 1. Pathways involving mammalian target of rapamycin (mTOR). Ovals with a slash indicate inhibitory pathways; the remaining pathways are stimulatory. RTK: receptor tyrosine kinase; GPCR: G protein-coupled receptor; RAS: oncogenic protein associated with membrane; P13K: phosphatidylinositol-3 kinase; PTEN: phosphatase and tensin homologue tumor suppressor; IKK: inhibitor of κB kinase; NF-κB: nuclear factor κB; PDK1: phosphoinositide-dependent protein kinase 1; TSC: tuberous sclerosis proteins; Akt: protein kinase B (a serine-threonine kinase); S6: S6 kinase; CCI-779, RAD001, and AP23573: rapamycin analogues; FKBP12: FK506 binding protein; mLST8: a protein of unknown function that associates with mTOR; CDKs: cyclin dependent kinases; 4E-BP: 4E binding protein; eIF: eukaryotic initiation factor.

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mTOR, which also is called FK506-binding protein (FKBP12), rapamycin-associated protein (FRAP), rapamycin and FKBP12 target (RAFT1), rapamycin target (RAPT1), and sirolimus effector protein (SEP), is a 289-kilodalton (kD) serine/threonine kinase orthologue of target of rapamycin 1 (TOR1) and TOR2 in Saccharomyces cerevisiae.1, 3–6 The mTOR gene maps to human chromosome 1p36.2.

Rapamycin (sirolimus), a macrocyclic lactone, was identified as an antifungal agent after its isolation from Streptomyces hygroscopicus in the middle 1970s.7–9 It is a structural analogue of tacrolimus (FK506). Rapamycin's antitumor activity was demonstrated in the 1980s in a National Cancer Institute screening program.10–12 The demonstration of rapamycin's antineoplastic properties led to the identification of mTOR as a potential target for chemotherapy. TOR originally was identified genetically by mutations in yeast that conferred resistance to the growth-inhibitory properties of the FKBP-rapamycin complex.13 The TOR1 and TOR2 genes encode two large and highly homologous proteins. The structurally and functionally conserved mammalian counterpart, mTOR, was discovered subsequently based on its FKBP-rapamycin binding properties.3–6

The mTOR protein contains multiple subdomains with sequence and positions that have been conserved highly throughout evolution. Human, mouse, and rat mTOR proteins share a 95% identity at the amino-acid level.1, 14, 15 This conservation suggests that these domains are essential for cellular functioning.

mTOR is comprised of up to 20 tandemly repeated motifs comprised of Huntington, elongation factor 3, the A subunit of protein phosphatase 2A (PP2A), and TOR (HEAT motifs) at the N-terminus; whereas the C-terminus contains an FRAP-ataxia telangiectasia-mutated, transformation/transcription domain-associated protein (FAT) domain; an FKBP12-rapamycin binding (FRB) domain; a catalytic kinase domain; a probable autoinhibitory or repressor domain; and an FAT carboxy-terminal domain (Fig. 2).16, 17 Rapamycin and its analogs bind to FKBP12. This complex binds to the FRB domain of mTOR and inhibits its kinase activity. Because the catalytic domain in the C-terminus of mTOR is highly homologous to the lipid kinase domain of phosphatidylinositol 3′ kinase (PI3K), mTOR is classified as a member of the PI3K-related protein kinase (PIKK) family.1, 14 PIKK are involved in many critical regulatory cell cycle functions pertaining to progression and checkpoints that govern cellular responses to DNA damage, DNA repair, and DNA recombination.18

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Figure 2. The structure of mammalian target of rapamycin (mTOR). Gray: The first 1200 amino acids comprise domains named for Huntington, elongation factor 3, the regulatory A subunit of protein phosphatase 2A, and Tor 1p (HEAT domains). This motif consists of stretches of ≈ 40 amino acids in at least 3 repeats and displays a consensus pattern of hydrophobic, proline, aspartatic acid, and arginine residues. Green: All phosphatidylinositol 3′ kinase (PI3K)-related protein kinase families (PIKKs) possess a short segment at their extreme carboxyl terminus, termed the rapamycin-associated protein (FRAP)-ataxia telangiectasia mutated (ATM)-transformation/transcription domain-associated protein (TRRAP), carboxy-terminal homology domain (FATC domain) and a region of weaker homology between amino acids 1382–1982 in the human FRAP/mTOR (FAT domain). Because the FAT domain always is found in combination with the FATC region, it has been postulated that intramolecular interactions between FAT and FATC modulate kinase activity. Red: The FK506 binding protein (FKBP12)/rapamycin-binding domain (FRB) lies immediately amino terminal to the kinase domain and downstream of the FAT domain. PKB: protein kinase B. Yellow: Catalytic domain.

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mTOR also is associated with a novel 150-kD peptide called regulatory-associated protein of mTOR (raptor). Raptor may act as a bridging protein that presents substrates to the mTOR kinase domain for optimal phosphorylation of downstream targets.19, 20 Raptor may serve as a scaffolding protein because, in addition to mTOR, it binds to p70 S6 kinase (p70S6K) and 4E-binding protein 1 (4E-BP1) through a TOR signaling motif (TOS).21 Another protein that associates with mTOR is mLST8, the function of which is unknown to date.19 The closely related yeast LST8 homologue, which negatively regulates RTG1/3 and GLN3 gene expression, thus limiting ketoglutarate, glutamate, and glutamine synthesis, is involved in the maintenance of cell wall integrity.22, 23 Kim et al. recently identified a protein named GbetaL that binds to the kinase domain of mTOR and stabilizes the interaction of raptor with mTOR.24 GbetaL participates in nutrient-mediated and growth factor-mediated signaling to S6K1, a downstream effector of mTOR, and in the control of cell size. The binding of GbetaL to mTOR strongly stimulates the kinase activity of mTOR toward S6K1 and 4E-BP1, an effect that is reversed by the stable interaction of raptor with mTOR.

The Role of mTOR in Cellular Signaling

  1. Top of page
  2. Abstract
  3. The Role of mTOR in Cellular Signaling
  4. Proteins in the Pathway Proximal to mTOR
  5. Proteins in the Pathway Distal to mTOR
  6. mTOR Signaling Pathway Dysregulation, Cell Transformation, and Antineoplastic Effects of mTOR Inhibition
  7. mTOR Inhibitors in Clinical Development in the Hematologic Malignancies
  8. CONCLUSIONS
  9. REFERENCES

Although all of the elements of the signal transduction pathways linked to mTOR have not been elucidated to date, a pathway that seems to be a key modulator involves PI3K/Akt (Fig. 1). PI3K and Akt lie upstream of mTOR and interact with growth factors and their receptors as well as other mitogenic stimuli. Also noteworthy is evidence suggesting that mTOR may control certain characteristics of malignant cells, such as anchorage.25 The interactions of other proximal activators that are regulated by nutrients and by adenosine triphosphate are less well characterized.

Proteins in the Pathway Proximal to mTOR

  1. Top of page
  2. Abstract
  3. The Role of mTOR in Cellular Signaling
  4. Proteins in the Pathway Proximal to mTOR
  5. Proteins in the Pathway Distal to mTOR
  6. mTOR Signaling Pathway Dysregulation, Cell Transformation, and Antineoplastic Effects of mTOR Inhibition
  7. mTOR Inhibitors in Clinical Development in the Hematologic Malignancies
  8. CONCLUSIONS
  9. REFERENCES

RAS

Ras is a centrally located guanine triphosphate (GTP)-binding protein with a number of downstream signal-cascade substrates. Signaling downstream of ras has been identified in cells through the mitogen/extracellular signal-related kinase (MEK/ERK), PI3K/Akt-mTOR/S6K, and nuclear factor κB pathways.26 Ras directly activates PI3K and PI3K activity, which results in Akt membrane localization, phosphorylation, and activation.27 Activation of the MEK/ERK and Akt pathways may lead to secondary activation of S6K. Although Ras is located in the cytoplasm, its activation requires translocation to the cytoplasmic side of the plasma membrane. This process is mediated through posttranslational modification with covalent attachment of a prenyl group (usually farnesyl) to Ras by the enzyme farnesyl transferase.28 The farnesylation of Ras is essential for its transforming ability. GTP-guanine diphosphate (GDP) exchange factors (guanine exchange factors) convert inactive Ras-GDP to active Ras-GTP.

PI3K

In mammalian cells, mTOR is activated as a result of ligand binding of various growth factors (e.g., insulin-like growth factor [IGF]) to their receptors and by signaling through integrins and chemokines through G-protein coupled receptors that cause activation of PI3K. Activated PI3K, in turn, catalyses the conversion of phosphatidylinositol (4,5)-biphosphate (PIP2) to phosphatidylinositol (3,4,5)-triphosphate (PIP3). PIP3 then binds to the pleckstrin homology domain of Akt, which causes its dimerization and exposes its catalytic site.25 Among the signaling pathways downstream of PI3K, the Akt pathway is of particular interest in neoplasia, because it is involved in both the inhibition of apoptosis and the promotion of cell proliferation by affecting the phosphorylation status of cell survival and apoptosis-inducing proteins (e.g., BAD).29 PI3K and subsequent mTOR signaling are required for activation of S6K1 and other downstream targets.

Akt

Also known as protein kinase B, Akt is a serine-threonine kinase that affects the phosphorylation state of mTOR either directly by phosphorylation or indirectly through the tuberous sclerosis complex (TSC1/TSC2), which acts as a modulator between PI3K and Akt.30–33

Mutations of the tumor suppressor PTEN gene, which encodes a lipid phosphatase that inhibits PI3K dependent activation of Akt, occur commonly in a wide variety of tumor types.34–36 PTEN has a major role in embryonic development, cell migration, and apoptosis.37 PTEN regulates major signal transduction pathways and effectively terminates PI3K-mediated signaling.38 PTEN mutation is associated with constitutive activation of the PI3K/Akt pathway, resulting in cancers that generally are resistant to apoptosis.

TSC1/TSC2

The TSC1/TSC2 complex comprises hamartin (TSC1) and tuberin (TSC2). These proteins form a physical and functional complex in vivo that binds and inhibits mTOR.31–33 Akt phosphorylates TSC2, which impairs inhibition of mTOR activity, possibly through dissociation of the TSC1/TSC2 complex. Loss of TSC1/TSC2 results in mTOR dependent increase in kinase activity of p70S6K, a serine-threonine kinase, and confers resistance of cells to amino-acid starvation.32 Conversely, coexpression of TSC1 and TSC2 inhibits activation of S6K1 in nutrient-deprived cells.33 Evidence that TSC1/TSC2 lies upstream of mTOR comes from the observation that inhibition on mTOR by rapamycin does not influence the phosphorylation of TSC2.32, 33 Recent evidence indicates that TSC1/TSC2 is a GTPase-activating protein of the small G-protein Rheb, which, in turn, may induce S6K and 4E-BP1 phosphorylation.39, 40 Overexpression of TSC1 and TSC2 inhibits Rheb-mediated S6K activation, but loss of function mutations of TSC1/TSC2 does not.

Proteins in the Pathway Distal to mTOR

  1. Top of page
  2. Abstract
  3. The Role of mTOR in Cellular Signaling
  4. Proteins in the Pathway Proximal to mTOR
  5. Proteins in the Pathway Distal to mTOR
  6. mTOR Signaling Pathway Dysregulation, Cell Transformation, and Antineoplastic Effects of mTOR Inhibition
  7. mTOR Inhibitors in Clinical Development in the Hematologic Malignancies
  8. CONCLUSIONS
  9. REFERENCES

Eukaryotric initiation factor 4F and 4E-BPs

A trimeric complex of eukaryotic initiation factors (eIFs), eIF4F is comprised of the cap-binding protein eIF4E, the scaffold protein eIF4G, and the RNA helicase eIF4A.1 The complex is necessary for recruitment of the ribosome to mRNA. This step also is called the initiation phase. It results in the positioning of a charged ribosome (80s ribosome loaded with tRNA) at an initiation codon. This recruitment process is the rate-limiting step in translation.1 The cap on the mRNA is recognized by the initiation factor eIF4E. In mammalian cells, changes in translation rates are correlated with changes in the level or activity of eIF4F, resulting in differences in the rate of ribosomal recruitment to mRNA, i.e., growing or stimulated cells contain high levels of eIF4F. Currently, it is unclear exactly how mTOR regulates S6K and 4E-BP activity: by direct phosphorylation, by activation of an intermediary kinase, and/or by inhibiting a phosphatase, such as PP2A. eIF-4E is important for efficient translation of RNA messages that contain complex secondary structures in the 5′-untranslated region, including growth factors and cell cycle regulators, such as cyclin D1.

Mammalian eIF4F formation is regulated by a family of translation repressors, the eIF4E binding proteins (4E-BPs).41 4E-BP1 is a low molecular-weight protein (also known as phosphorylated heat-stable and acid-stable protein 1 [PHAS-1]) that binds to eIF4E depending on the phosphorylation status of 4E-BP. In its unphosphorylated state, which predominates in relatively quiescent cells, and under growth factor-deprived conditions, 4E-BP binds avidly to eIF4E, which inhibits its activity and, hence, protein translation.42 In response to proliferative stimuli initiated by growth factors, hormones, mitogens, cytokines, G protein-coupled agonists, and integrins, 4E-BP1 is phosphorylated by mTOR and by other kinases that decrease its affinity for eIF4E. This then leaves the eIF4F complex free for initiation of protein translation.43–45 MTOR also may dephosphorylate 4E-BP1 indirectly using other phosphatases.46, 47 Because the eIF4E pathway is required for translation of mRNAs encoding cyclin D1, ornithine decarboxylase44, 48, 49 inhibition of mTOR leads to slowing or arrest of cells in the G1-phase of the cell cycle. This inhibition results in deficiency of active cyclin dependent kinase 4 (CDK4)/cyclin D1 complexes required for retinoblastoma protein (pRb) phosphorylation. In addition, rapamycin blocks elimination of CDK inhibitor p27 and facilitates the formation of cyclin/CDK-p27 complexes.50, 51 Rapamycin also up-regulates p27 at the mRNA and protein levels and inhibits cyclin-A dependent kinase activity in exponentially growing cells.48, 52 This may explain in part the profound inhibition of G1-phase to S-phase transition observed after rapamycin treatment.

S6K

The serine-threonine kinase S6K is another important downstream target on the mTOR pathway. Both S6K and 4E-BP1 contain consensus TOS motifs in the N-terminus and C-terminus, respectively, that are required for phosphorylation and regulation of their activity by mTOR.53 In addition, 4E-BP1 requires the presence of an RAIP motif in the N-terminus for phosphorylation of additional sites by mTOR to occur.54 In the presence of proliferative stimuli, mTOR phosphorylates and, thus, activates S6K. S6K, in turn, phosphorylates the 40s ribosomal protein S6.55, 56 This leads to active translation of mRNAs with a 5′-terminal oligopyrimidine. Inactivation of S6K decreases synthesis of ribosomal proteins and components of the translational machinery (e.g., elongation factors).57, 58 Activation of S6K is complex. The process is mediated by multiple upstream kinases, including mTOR.59 Phosphoinositide dependent protein kinase 1 (PDK1) is one such kinase involved in phosphorylation of S6K.60

RNA polymerases I and III

Rapamycin also inhibits the function of RNA polymerase I (Pol I) and Pol III—the latter controls 5S and tRNA transcription.61, 62 Consistent with this, it has been shown that the functions of Pol I and Pol III are TOR dependent; and in yeast, but not in mammalian cells, Pol I and Pol III utilize PP2A.63 The control of RNA Pol I and Pol III function by mTOR may occur through regulation of pRB, because pRB phosphorylation and inactivation are blocked by treatment with rapamycin.48

Signal transducers and activators of transcription 3

Recent data have indicated significant interactions between mTOR and signal transducers and activators of transcription 3 (STAT3).64 STAT3 mediates up-regulation of c-myc and stabilizes cyclin D. mTOR directly phosphorylates and activates STAT3 in vitro, thus leading to transcription of STAT3-responsive genes, and rapamycin inhibits STAT3 activation.64

mTOR Signaling Pathway Dysregulation, Cell Transformation, and Antineoplastic Effects of mTOR Inhibition

  1. Top of page
  2. Abstract
  3. The Role of mTOR in Cellular Signaling
  4. Proteins in the Pathway Proximal to mTOR
  5. Proteins in the Pathway Distal to mTOR
  6. mTOR Signaling Pathway Dysregulation, Cell Transformation, and Antineoplastic Effects of mTOR Inhibition
  7. mTOR Inhibitors in Clinical Development in the Hematologic Malignancies
  8. CONCLUSIONS
  9. REFERENCES

Based on numerous lines of preclinical research, interest in mTOR inhibition in the hematologic malignancies is increasing. Although to our knowledge no mutations in mTOR have been detected in tumors to date, signaling through mTOR appears to be pivotal in tumor growth.1 Consistent with the critical importance of PI3K and mTOR signaling in the control of growth and proliferation, dysregulation of the activity of several components of this pathway is associated with cellular transformation. PI3K and Akt are considered protooncogenes, and their pathways may be inhibited by the tumor suppressor gene PTEN.65 Akt overexpression transforms mammalian cells in culture.66 PI3K activity is up-regulated in many cancer cells.67 Tumors that rely on paracrine or autocrine stimulation of receptors that constitutively stimulate the PI3K/Akt/mTOR pathway or tumors with mutations that activate the PI3K/Akt signal transduction pathway may depend on rapamycin-sensitive pathways for growth and, thus, may be particularly sensitive to rapamycin analogs. PTEN status in tumor cells, therefore, may be an important predictor of sensitivity to rapamycin analogs.68 An important issue in future studies will be the predictive ability of PTEN status for a clinical response to mTOR inhibitors.69 Up-regulation of PI3K activity or increased levels of phosphorylated Akt in the absence of PTEN mutations are predictive of response to mTOR inhibitors.70, 71

Oncoproteins derived from PI3K itself have been identified. p65-PI3K, a truncation mutant of the regulatory PI3K p85 subunit, was isolated from a thymic lymphoma.72 This protein drove constitutive PI3K activation, causing lymphoproliferative disorders and autoimmunity when expressed in a transgenic murine model of T lymphocytes.73 Malignant lymphoid cells may depend on activation of the pathway through cytokine activation of PI3K or Akt or through loss of PTEN, which would augment the activity of mTOR. Thus, the activation of the PI3k/Akt pathway seems to be important to normal and neoplastic T-cell and B-cell proliferation.74, 75 Overexpression of eIF4E or eIF4G reportedly o results in malignant transformation of fibroblasts.76 Elevated levels of eIF4F components have been detected in a variety of tumors, including non-Hodgkin lymphomas.1 Mantle cell lymphoma (MCL) with t(11;14)(q13;q32) translocation is associated with cyclin D1 overexpression and a poor prognosis. It has been shown, as discussed earlier, that rapamycin inhibits the expression of cyclin D1. Cell lines with PTEN mutations are extraordinarily sensitive to rapamycin.77 In addition, abnormalities of the G1 checkpoint, such as pRb, p27, and cyclin D1, also may increase the sensitivity of tumors to rapamycins.78 This proposes exciting therapeutic options for patients with MCL who have such these defects. The National Cancer Institute is sponsoring a Phase II trial in patients with this disease.

Cytokines, including interleukin-6 and IGF-1, support the growth and prevent the apoptosis of malignant plasma cells.79–83 In the bone marrow, it has been shown that cytokines activate PI3K in myeloma cells.84 Constitutive activation of the Akt pathway has been described in multiple myeloma cell lines, and persistent activation may be important in myeloma cell expansion.85, 86 Multiple pathways that contribute to the stimulation of cytokine independent growth are activated downstream of RAS in multiple myeloma cells, suggesting that therapeutic strategies that target these pathways may be efficacious in myeloma cells with RAS mutations.26 Ras mutations may occur in up to 30% of patients with acute myeloid leukemia (AML), and initial data indicate that farnesyl transferase inhibitors, which may affect Ras activity, have significant activity in the myeloid leukemias.87

AML blasts and myeloma cells have demonstrated constitutive activation of the PI3K pathway, and PI3K appears to be necessary for their survival.83, 88 It has been shown that PI3K inhibitors induce apoptosis in such cells and in cells from patients with bcr-abl positive malignancies. Cells that express bcr-abl have up-regulated PI3K/Akt pathways, which are essential for proliferation.89 It has been shown that treatment with rapamycin lowers bcr-abl levels and induces apoptosis of K562 cells, whereas exposure to higher doses for prolonged periods results in erythroid differentiation.90

Rapamycin effectively induces granulocytic differentiation of human myeloid leukemic HL-60 and ML-1 cells.90 It has been shown that rapamycin inhibits BCR-ABL-induced vascular endothelial growth factor expression and hypoxia-inducible factor-1 expression in growth factor dependent Ba/F3 cells.91 The stem cell myeloproliferative disorder associated with t(6;8)(q27;p12) is dependent on the mTOR pathway for survival.92 Rapamycin also induces cell cycle arrest in certain B-chronic lymphocytic leukemia cells by inhibiting phosphorylation of S6K.93

mTOR Inhibitors in Clinical Development in the Hematologic Malignancies

  1. Top of page
  2. Abstract
  3. The Role of mTOR in Cellular Signaling
  4. Proteins in the Pathway Proximal to mTOR
  5. Proteins in the Pathway Distal to mTOR
  6. mTOR Signaling Pathway Dysregulation, Cell Transformation, and Antineoplastic Effects of mTOR Inhibition
  7. mTOR Inhibitors in Clinical Development in the Hematologic Malignancies
  8. CONCLUSIONS
  9. REFERENCES

CCI-779 (sirolimus).

CCI-779 (sirolimus) is a rapamycin analog that specifically inhibits mTOR.16, 25, 26, 71, 77, 84, 94–102 CCI-779 inhibits the growth of a wide range of histologically diverse tumor cells. Cell lines with the greatest sensitivity to this agent include central nervous system carcinoma, leukemia (T-cell), breast carcinoma, prostate carcinoma, and melanoma. CCI-779 is being developed as a cytostatic agent to delay the time to tumor recurrence/progression or to increase survival in patients with various malignancies. Studies in PTEN-deficient myeloma cell lines exhibit marked sensitivity to G1 arrest (50% inhibitory concentration [IC50] < 1 nM) after treatment with CCI-779, whereas myeloma cells containing wild type PTEN are at least 1000-fold less sensitive.103 These data suggest that the identification of PTEN mutations within tumor cells may be predictive of sensitivity to CCI-779 therapy.69, 71

In studies published to date, acute hypersensitivity reactions that begin shortly after the start of intravenous (IV) infusion (usually, but not always, with the first infusion) and that end after the infusions stop have been reported with CCI-779 administration. Among patients who underwent organ transplantation and received sirolimus, pneumonitis/pulmonary infiltrates and alveolitis have been reported in those who received IV CCI-779. Some patients have been asymptomatic, with pneumonitis detected on computed tomography scans or chest X-rays; whereas other patients have had symptoms of dyspnea, cough, and fever. These symptomatic patients had CCI-779 discontinued and received corticosteriods and/or antibiotics with improvement. There have been a few instances of recurrence of symptoms/signs of pneumonitis with CCI-779 retreatment. Increased fibrinogen levels have been reported in some patients who received CCI-779. The clinical significance of this event is not certain. To date, there does not appear to be an increased incidence of venous or arterial thrombosis in patients on CCI-779 studies. CCI-779 and rapamycin are metabolized primarily by CYP3A4 in human liver microsomes—this potential for drug-drug interactions will be assessed in on-going studies. In studies published to date, with weekly doses ranging from 25 mg to 250 mg of CCI-779 IV, commonly reported, drug-related, adverse events include rash, mucositis, asthenia, nausea, and acne.104 Because current data indicate that sustained mTOR inhibition is associated with a weekly 25-mg intravenous dose of CCI-779, this regimen is being investigated in patients with refractory hematologic malignances.

RAD001C (everolimus)

RAD001 (everolimus) also is a rapamycin analog that is being developed as an antiproliferative agent—it has been approved in Europe as an immunosuppressant agent in the solid organ transplantation setting.16, 25, 105–108 RAD001 exerts its activity on interleukin dependent and growth factor dependent proliferation of cells through its high affinity for an intracellular receptor protein, the immunophilin FKBP-12. The resulting FKBP-12/RAD001 complex then binds with mTOR to inhibit downstream signaling events. In vitro studies have shown that RAD001 can inhibit the proliferation of numerous cell lines originating from solid tumors, with the most sensitive cell lines showing IC50 values at the nanomolar level. In addition, experiments in vitro with human umbilical endothelial cells and in animal models of angiogenesis suggest an additional antiangiogenic effect, presumably through mTOR inhibition in proliferating endothelial cells.

Excessive growth of Epstein–Barr virus (EBV)-transformed B lymphocytes often is the cause of life-threatening posttransplantation lymphoproliferative disorders (PTLDs).109 RAD001 has an antiproliferative effect on EBV-transformed B-cells in culture and in a mouse model, blocking these cells in G1-phase and inducing apoptosis.107 Reports of three patients who had PTLDs and were treated successfully with rapamycin and rituximab have been published.110, 111 These data suggest that rapamycin or its analogs should be investigated further in patients with PTLDs. Xu et al. recently reported on in vitro studies of RAD001 in patients with AML.88 Those authors confirmed the activation of PI3K and Akt in AML blasts and also showed S6K and 4EBP activation. Incubation of AML blasts with RAD001 as a single agent induced a small decrease in survival of AML blasts.

There are extensive safety data regarding RAD001 because of the advanced stage of its development in solid organ transplantation.112–114 Safety data include single-dose studies in nontransplantion patients and short-term and long-term studies in transplantation patients who received daily RAD001 as a part of an immunosuppressant, multidrug regimen that consistently included cyclosporin A, glucocorticoids, and occasionally other drugs (azathioprine, basiliximab). Controlled studies in the transplantation setting consisted of dosage comparisons and comparisons between RAD001 and mycophenolate mofetil or azathioprine as additions to cyclosporin and steroids. RAD001 is tolerated very well, with mild-to-moderate, suspected adverse drug reactions, mainly headache. Daily treatment with RAD001 has been associated with decreases in absolute neutrophil counts and platelet counts that rarely lead to severe leukopenia or thrombocytopenia. Chronic treatment with daily RAD001 also has been associated with an increase in serum lipid levels; and long-term treatment with RAD001 has been associated with reduced testosterone levels, probably the result of interference with sterol metabolism, which does not translate into increased sexual dysfunction. A dose-finding study of RAD001 in patients with refractory hematologic malignancies is being planned.

AP23573

AP23573 is a nonprodrug rapamycin analog, small-molecule inhibitor of mTOR.100 Phase I studies of this compound in patients with solid tumors have begun recently—studies in patients with a range of refractory hematologic malignancies are being planned.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. The Role of mTOR in Cellular Signaling
  4. Proteins in the Pathway Proximal to mTOR
  5. Proteins in the Pathway Distal to mTOR
  6. mTOR Signaling Pathway Dysregulation, Cell Transformation, and Antineoplastic Effects of mTOR Inhibition
  7. mTOR Inhibitors in Clinical Development in the Hematologic Malignancies
  8. CONCLUSIONS
  9. REFERENCES

The central role of mTOR in many critical cellular processes makes it an attractive target in patients with hematologic malignancies. Many critical issues remain to be addressed. Which, if any, of the rapamycin analogs will have significant clinical activity? Will the future role of these analogs, as presumed cytostatic agents, be in combination with cytotoxic or other targeted therapies? Will PTEN deficiency predict for responsive patient subsets?69 What are the baseline or consequent responses in the PI3K/Akt pathways that may predict for response or that may provide a rational basis for future combination therapies? What are the critical factors that cause apoptosis in a malignant cell exposed to an mTOR inhibitor?16, 25, 99, 115 What are the roles of IGF-1, Ask1, c-Jun, and JNK in such terminal pathways?116 The answers to these questions will assist in the development of novel therapeutic agents for patients with hematologic disorders.

REFERENCES

  1. Top of page
  2. Abstract
  3. The Role of mTOR in Cellular Signaling
  4. Proteins in the Pathway Proximal to mTOR
  5. Proteins in the Pathway Distal to mTOR
  6. mTOR Signaling Pathway Dysregulation, Cell Transformation, and Antineoplastic Effects of mTOR Inhibition
  7. mTOR Inhibitors in Clinical Development in the Hematologic Malignancies
  8. CONCLUSIONS
  9. REFERENCES