Mammalian Target of Rapamycin as a Target in Hematological Malignancies

Mammalian Target of Rapamycin as a Target in Hematological Malignancies he era of targeted therapy is here. Understanding the regulation of cellular protein synthesis is crucial in the development of novel agents that target certain pathways causing smarter cell kill. The mammalian target of rapamycin (mTOR) role in cellular protein synthesis affects different aspects of the cell growth, including differentiation, cell cycle progression, protein degradation, and apoptosis, as well as protein kinase C signaling and transcription.1-5 mTOR has other known names, including rapamycin-associated protein (FRAP), FK506-binding protein (FKBP12), rapamycin and FKBP12 target (RAFT1), rapamycin target (RAPT1), and sirolimus effector protein (SEP). It is a 290-kDa serine–threonine kinase orthologue of target of rapamycin 1 (TOR1) and target of rapamycin 2 (TOR2) in Saccharomyces cerevisia.6-10 The TOR1 and TOR2 genes encode two large, highly homologous proteins. The mTOR gene maps to chromosome 1p36.2 in humans. Human, mouse, and rat mTOR proteins share a 95% identity at the amino acid level,6,11,12 suggesting that these domains are critical for cellular function. Rapamycin (sirolimus) is a macrocyclic lactone that is naturally produced by Streptomyces hygroscopicus bacteria. It was initially identified as an antifungal agent.13-15 It was also used as an immunosuppressant agent for prevention of rejection after solid organ transplantation16,17 and in rheumatoid arthritis given its effect on lymphocyte proliferation and cytokine production, which affect the pathogenesis of rheumatoid arthritis significantly.18 It is widely used currently in drug-eluting stents to prevent restenosis given its antiproliferative effect.19 The demonstration of rapamycin’s antitumor properties was initially reported in the National Cancer Institute (NCI) screening program in the 1980s.20-22 This review will focus on the role of mTOR as a target in hematological malignancies.

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mTOR: Structure and Function mTOR is formed of up to 20 tandemly repeated motifs of HEAT (Huntington, EF3, A subunit of PP2A, and TOR) at the N terminus. The C terminus, on the other hand, contains: a FRAP–ataxia telangiectasiamutated, Transformation/transcription domain-associated protein (FAT) domain, a FKBP12–rapamycin binding (FRB) domain, a probable autoinhibitory or repressor domain, a catalytic kinase domain, and a FAT carboxy-terminal (FATC) domain.23,24 Given that the catalytic domain in the C terminus of mTOR is very similar to the lipid kinase domain of phosphatidylinositol 3=kinase (PI3K), mTOR is considered a member of the PI3K-related protein kinase family (PIKK).6,11 It is frequently referred to as the Pl3K/Akt/mTOR pathway, and it is critical in PIKKs regulating cellular responses to DNA damage, repair, and recombination.25,26 Rapamycin and its analogs bind to FKBP12, creating a complex which binds to the FRB domain of mTOR and inhibits its kinase activity.23 mTOR is functionally composed of two complexes: mTOR Complex 1 (mTORC1) and mTORC2.27 mTORC1 is composed of mTOR, a novel 150-kDa peptide regulatory associated protein of mTOR (raptor) and mammalian LST8/G-protein ␤-subunit like protein (mLST8/G␤L).28-30 Raptor acts by presenting downstream target substrates to the mTOR kinase domain for phosphorylation, and it also binds to its targets p70S6K and 4E-BP1 via TOR signaling motif (TOS).31-34 The mLST8 protein also associates with mTOR, the yeast LST8 homologue, which negatively regulates RTG1/3 and GLN3 gene expression resulting in limiting ketoglutarate, glutamate, and glutamine synthesis, which is involved in the maintenance of cell wall integrity.24,30-32 G␤L binds to the mTOR kinase domain and stabilizes its interaction with mTOR.33 Raptor and mTOR share a strong interaction at the N terminus but a weak one at the C terminus. mTORC1 is activated in the presence of nutrients through significant weakness of the C terminus, leading to mTOR kinase activation. The C terminus is strengthened during starvation status, leading to deactivation of mTOR.29 On the other hand, mTORC2 is composed of mTOR, a companion of mTOR (rictor), G␤L, and mammalian stressactivated protein kinase interacting protein 1 (mSin1).35 The rictor is insensitive to rapamycin, leading to the fact that the mTOR sensitivity to rapamycin is exerted through mTORC1. mTORC2 stimulates Akt phosphorylation at threonine308 by the phosphoinositide-dependent kinase 1 (PDK1), leading to Akt activation.36 162

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AKT/mTOR Pathway in Cancer The activation of mTOR has been implicated in many human malignancies,37-52 and it has also been described in the spread of cancer via lymphatics. Rapamycin has been shown in in vivo experimentation to decrease the number and lymphatic vessels in the primary tumors revealing anti-lymphangiogenic effect.53,54 An interesting concept is its role in minimal residual disease, which is surgical margins that are free from tumor but over-express eIF4E. This has been reported in head and neck cancer with rapamycin given as adjuvant therapy yielding survival advantage.55 Endothelial growth factor receptor (EGFR) regulates PI3K/ Akt pathway. Activation of Akt leads to the stimulation of antiapoptotic pathways, promoting cell survival. The activity of the EGFR inhibitor erlotinib in non-small-cell lung cancer is dependent on inhibition of the PI3K phosphoinositide-dependent kinase 1-Akt-mTOR pathway. Rapamycin inhibited significantly S6 in all cell lines derived for different tissue types (non-small-cell lung, pancreatic, colon, and breast), and this was accompanied by activation of Akt phosphorylation. Erlotinib only inhibited extracellular signal-regulated kinase, Akt, and S6 in most sensitive cell lines. The combination of rapamycin and erlotinib downregulated rapamycin-stimulated Akt activity, so adding rapamycin to erlotinib could improve its activity.56 A phase II study of a combination of temsirolimus or everolimus with letrozole demonstrated a better progression-free survival in the combination arm than in the letrozole alone arm.57 Myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML) are believed to be two spectrums of the same disease process. The activation of mTOR pathway has been shown in both diseases.58-60 This pathway has also been described in diffuse large B-cell lymphoma61 and anaplastic large cell lymphoma.62 Mantle cell lymphoma is characterized by an over-expression of cyclin D1 resulting from chromosomal translocation (t11,14). The drug suberoylanilide hydroxamic acid (SAHA; vorinostat) was shown to decrease the level of cyclin D1 by 90% through the inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR/ eIF4E-BP pathway.63

Side Effects of mTOR Inhibitors A meta-analysis to study the effect of TOR-1 inhibitors (sirolimus and everolimus) on male gonads showed that these agents have a negative effect on spermatogenesis as evidenced by the decrease in testosterone level and the increase in LH and FSH.64 Oligospermia has been Curr Probl Cancer, July/August 2008

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implicated in sirolimus-associated infertility.65 Although preclinical studies on animal models showed sirolimus to be a potentially non-nephrotoxic drug, clinical data reported nephrotoxicity to be a concern.66 Sirolimus has been reported to be associated with proteinuria and acute renal failure, especially in patients with pre-existing renal damage. Sirolimus-induced nephrotoxicity is mostly mulifactorial but may involve glomerular injury and suppression of compensatory renal cell proliferation and repair processes. It is advised to monitor patients for occurrence of proteinuria and renal dysfunction. Like in diabetic nephropathy, use of angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers may alleviate proteinuria and kidney damage.67 Temsirolimus and its metabolite, sirolimus, are cytochrome P450 (CYP) 3A4/5 substrates resulting in potential interaction with drugs that induce CYP3A activity.68 A retrospective study evaluated pulmonary side effects of temsirolimus. Medical records of 22 patients who were treated with weekly doses of temsirolimus 25 mg were reviewed. Eight patients (36%) developed drug-induced pneumonitis. Two different patterns were described on imaging: ground glass opacities and lung parenchymal consolidation. The interesting finding of this study was that half of these patients had symptoms, with dyspnea and dry cough being the most common, but the other half were asymptomatic, raising the possibility of the role of screening imaging in patients receiving temsirolimus.69 There seems to be a difference in the side effects on lipid profile between the mTOR inhibitors. Sirolimus was found to increase serum triglyceride and total cholesterol concentrations in contrast to everolimus, which increased HDL concentration in heart transplant recipients with renal insufficiency. This resulted in increased frequency of statin use in the sirolimus group. Both drugs resulted in reliable immunosuppression in this setting.70

Resistance to mTOR Inhibitors With the growing role of mTOR inhibitors in cancer treatment, resistance is a concern. Several mechanisms have been reported, although their interaction is complex and still a lot remains unknown. Mutations in FKBP12 prevent rapamycin from binding to mTOR, leading to rapamycin resistance.71 Defects in mTOR-regulated proteins, like 4E-BP1, PP2Arelated phosphatases, and p27(Kip1), cause rapamycin insensitivity, hence resistance.72,73 Cancer cell lines with intrinsic rapamycin resistance were found to have low 4E-BP:eIF4E ratios.74 Point mutations in S6K1 affect rapamycin sensitivity in vitro.75,76 P53 status has been influential in 164

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rapamycin resistance as well.77 Mutated ataxia telangiectasia cells are rapamycin-resistant. Defects in signaling could lead to resistance, but additional mutations could also play a role.78,79 Other factors have been implicated as well such as 14-3-3 proteins80 and PTEN/Akt status.81 Mutation in the FRB domain of mTOR has been implicated in high-level resistance.82 PIM-2 is a key component in hematopoietic cells growth and survival, and its overexpression has been implicated in developing resistance to rapamycin.83

mTOR in Specific Hematolgical Malignancies Malignancies with PTEN mutations are resistant to apoptosis but could be especially sensitive to mTOR inhibitors. A study by Shi et al.84 demonstrated that myeloma cells that are deficient in PTEN were not apoptosis-resistant. But these particular cells were significantly more sensitive to CCI-779 (sirilomus), which was the mTOR inhibitor the authors used in their experiment.84

Mantle Cell Lymphoma Mantle cell lymphoma (MCL) is characterized by t(11;14) and cyclin D1 overexpression. Through immunohistochemical methods and tissue microarray, it has been proved that mTOR signaling pathway is activated in this disease and it contributes to tumor cell survival.85 MCL has especially poor prognosis, and it is an attractive area for testing new targeted therapy. Several agents are currently being tested in this disease.86 Rapamycin has been shown in vivo to inhibit proliferation in three MCL cell lines, resulting in reduction of cyclin D3 expression with no effect on cyclin D1 levels. This finding was duplicated in cells from a MCL patient.87 This led to granting an FDA approval for the use of sirolimus in this disease.

Multiple Myeloma Activation of adenosine monophosphate activated protein kinase inhibits mTOR and P70S6 kinase (P70S6K) as well as AKT phosphorylation in multiple myeloma (MM) cell lines. It induced S-phase cell cycle arrest. This inhibition persisted in the face of adding IL-6, IGF-1, and HS-5 stromal cell-conditioned medium.88 Rapamycin also induces G0/G1 arrest in MM cell lines, leading to increase in the cyclin-dependent kinase inhibitor p27 and a decrease in cyclins D2 and D3. Rapamycin induced apoptosis and it sensitized MM cells and cell lines to dexamethasoneinduced apoptosis despite adding the antiapoptotic agents insulin growth factor-I (IGF-I) and interleukin 6 (IL-6). This effect was associated with Curr Probl Cancer, July/August 2008

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a decreased expression of cyclin D2 and surviving.89 Immunoblot assays had shown that rapamycin inhibited expressions of XIAP, CIAP1, HSP-27, and BAG-3, which may explain what has been noticed about rapamycin sensitizing multiple myeloma cells to dexamethasone-induced apoptosis.90 This suggests that the combination of rapamycin and dexamethasone is active in vivo in MM. Inhibitors of HSP90 protein are active in vitro and in vivo in MM. The combination of these agents with mTOR inhibitors have been shown to have synergistic activity in MM cell lines through induction of apoptosis and cell cycle arrest. Both groups of inhibitors inhibit osteoclast cells affecting the microenvironment of the bone marrow, which is especially attractive in the treatment of MM.91

Acute Leukemia PI3k/Akt pathway is involved in the pathophysiology of childhood acute lymphoblastic leukemia (ALL) and it has a role in blast survival. Avellino et al.92 showed rapamycin to induce apoptosis of blasts in 56% of samples of the bone marrow. Rapamycin also increased doxorubicininduced apoptosis, even in non-responder samples. This is probably explained by their finding that FKBP51, an immunophilin inhibited by rapamycin, is essential for drug-induced nuclear factor ␬B (NF-␬B) activation in leukemia. Anthracyclines activate NF-␬B, and rapamycin inhibited doxorubicin-induced NF-␬B. Interestingly, rapamycin did not increase doxorubicin-induced apoptosis when NF-␬B was overexpressed. NF-␬B pathway through FKBP51 is a newly discovered pathway that rapamycin can target along with the previously described PI3k/Akt pathway leading to therapeutic benefit in ALL.92 Rapamycin is an emerging treatment in ALL,93 and its activity as a single agent in B-precursor ALL cell lines has been documented. It caused apoptotic cell death which was reversible by adding IL-7. This is possibly due to p70S6 kinase being regulated by both rapamycin and IL-7. E mu-ret transgenic mice with advanced disease treated with daily rapamycin as a single agent more than doubled their survival as compared with symptomatic controls.94 All-trans-retinoic acid (RA) is an active agent in acute promyelocytic leukemia (APL). RA treatment of primary APL blasts and RA-sensitive NB-4 cells resulted in phosphorylation/activation of mTOR and downstream induction of p70S6 kinase activity. Interestingly, this was not the case in NB-4 variant cell line (NB-4.007/6) that is resistant to the biologic effects of RA. RA also resulted in phosphorylation of the 4E-BP1 repressor of mRNA translation through mTOR pathway, causing its 166

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deactivation and dissociation from the eukaryotic initiation factor-4E (eIF-4E) complex.95 PI3-kinase-Akt-mTOR pathway has been shown to be activated in up to 60% of AML patient cells.96 In a series of 23 AML cases, rapamycin inhibited the growth of the most immature AML cell lines through G0/G1 cell cycle arrest. Rapamycin spared normal hematopoietic progenitors. It induced significant clinical responses in 4 of 9 patients with AML (either refractory/relapsed de novo or secondary), making it an attractive agent in this disease.97,98 It has been hypothesized that inhibition of both the mTOR pathway and glycolysis can work synergistically to affect the energy status in cancer cells. Combining rapamycin and glycolytic inhibitor produced synergistic apoptosis and cell growth inhibition in human lymphoma cells and leukemia cells. This combination suppressed glucose uptake and critically depleted cellular ATP pools, leading to cell death. No cytotoxicity was observed in vitro when rapamycin was combined with cytarabine.99

Chronic Myelogenous Leukemia Ribosomal protein S6 and 4E-BP1 are phosphorylated in chronic myelogenous leukemia (CML) cells. This phosphorylation is downstream of Bcr-Abl kinase and mTOR, meaning that mTOR is activated in CML. Rapamycin was found to potentiate arsenic trioxide-mediated suppression of primitive leukemic progenitors taken from the bone marrow of patients with CML.100 Rapamycin was also shown to enhance imatinib-mediated killing of CML cell lines in vitro, and it overcame imatinib resistance.101 Resistance to tyrosine kinases is unfortunate given their marked activity in this disease. BCR/ABL-kinase mutations are mostly to blame. Burchert et al.102 showed that Imatinib treatment activated the PI3K/Akt/mTOR pathway in BCR/ABL-positive LAMA-cells and primary leukemia cells in vitro, as well as in a chronic phase CML patient in vivo. In the early phase of imatinib resistance, PI3K/Akt-activation mediated survival before the kinase mutation manifested. Patients who were imatinibresistant or refractory had patterns of Akt-pathway activation independent of the kinase mutation. mTOR-inhibition overcame imatinib resistance only if Akt was strongly activated. This gives rationale to test combinations of imatinib and mTOR inhibitors in patients with positive BCR/ ABL.102 Rapamycin inhibited growth of CML cells of both imatinibresponsive or imatinib-resistant nature. This effect was also shown in an imatinib-resistant positive BCR-ABL leukemic patient. This was associated with G1 cell cycle arrest, induction of apoptosis, and downregulation of expression of vascular endothelial growth factor (VEGF). Curr Probl Cancer, July/August 2008

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Exogenously added VEGF did not correct the rapamycin-induced decrease in proliferation.103 Like imatinib, interferon has also been shown to activate mTOR in BCR-ABL positive cells. It resulted in downstream activation of p70S6 kinase. Imatinib, on the other hand, suppressed p70S6 kinase activity, consistent with inhibition of BCR-ABL-mediated activation of the mTOR/p70 S6 kinase pathway. Rapamycin had a synergistic effect with imatinib on primary leukemic granulocyte macrophage– colony-forming unit (CFU-GM) progenitors from patients with CML.104

Lymphoma Everolimus has antiproliferative activity in vitro and in NOD/SCID mice in vivo against Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) cells. It down-regulated the truncated isoform of the transcription factor CCAAT enhancer binding protein ␤ (C/EBP␤), leading to a disruption in the terminal differentiation with a transformed phenotype. Everolimus also inhibited NF-␬B activity, which is a critical survival factor of HL cells.105 Rapamycin is also effective in B-Chronic lymphocytic leukemia (B-CLL).106 mTOR is activated in follicular lymphoma (FL) through Syk. Inhibition of Syk by siRNA plasmids or piceatannol results in mTOR inhibition in FL cells. This effect was also seen in other lymphomas like mantle-cell and diffuse-large B-cell. Targeting Syk-mTOR pathway is a possible new target in the treatment of these disorders.107

Novel mTOR Inhibitors Sirolimus (CCI-779) Sirolimis is the most extensively studied mTOR inhibitor and the most widely used one in clinical practice. A phase I trial established the safety of sirolimus (CCI-779) given weekly over 30 minutes at doses of 25, 75, and 250 mg/m2. The most common toxicities were maculopapular rash, mucositis, and stomatitis, and they were reversible. Thrombocytopenia was the main dose-limiting toxicity but was also reversible. The maximum tolerated dose was not reached. No immunosuppression was observed even at the highest dose.108 Another phase I study reported the maximum tolerated dose to be 15 mg/m2/day for patients with extensive prior treatment and 19 mg/m2/day for patients who were minimally pretreated. Terminal half-life in that study was 13 to 25 hours.109 In phase II testing, the side effect profile was similar with maculopapular rash (76%), mucositis (70%), asthenia (50%), and nausea (43%). The most frequently occurring grade 3 or 4 adverse 168

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events were hyperglycemia (17%), hypophosphatemia (13%), anemia (9%), and hypertriglyceridemia (6%).110 It is known that CCI-779 inhibits the kinase activity of mTOR, resulting in inhibition of the mTOR-regulated translational controllers p70(s6) kinase and 4E-BP1. This led to postulate that measuring p70(s6) kinase activity may mirror the effect of sirolimus on cells. It was shown that the activity of inhibition of p70(s6) kinase is identical between peripheral blood mononuclear cells and cells from the tumor tissue. This makes the ease of measurement of the assay of p70(s6) kinase from peripheral blood an attractive tool in judging biochemical response to sirolimus.111 Witzig et al.112 treated 35 patients with relapsed or refractory mantle cell lymphoma with temsirolimus 250 mg intravenously every week as a single agent. The median age was 70 years and 91% of patients had stage 4. The overall response rate was an impressive 38% with the response lasting for 6.9 months. The most common toxicities were hematological with thrombocytopenia being the most dose-limiting side effect, although it was reversible.112 It is FDA approved in mantle cell lymphoma.

Everolimus (RAD001) Everolimus is an immunosuppressive macrolide with a stable 2-hydroxyethyl chain substitution at position 40 on the rapamycin structure. It was developed to be orally bioavailable more than sirolimus. It is currently the only mTOR inhibitor in oral form. It acts by blocking growth-driven transduction signals in the T-cell response to alloantigen so its site of action is at a later stage than tacrolimus. It has a high affinity for the intracellular receptor protein FKBP-12 through which it affects interleukin and growth-factor-dependent proliferation of cells. In vitro experiments with human umbilical endothelial cells (HUVECS) and animal models revealed a possible antiangiogenic effect of everolimus, possibly through inhibition of proliferating endothelial cells.113 Everolimus was developed as an immunosuppressive agent and is approved in Europe for that purpose in the setting of solid organ transplantation.114-118 Oral everolimus is readily bioavailable, and it reaches peak concentration after 1.3–1.8 hours. It is metabolized by the cytochrome P450 (CYP) 3A4, 3A5, and 2C8. Dosage should be reduced by half in patients with hepatic impairment.119 It was studied in a phase I/II study in relapsed or refractory hematologic malignancies (9 acute myelogenous leukemia, 5 myelodysplastic syndrome, 6 B-chronic lymphocytic leukemia, 4 mantle cell lymphoma, 1 of each: myelofibrosis, natural killer cell/T-cell leukemia, and T-cell prolymphocytic leukemia). The phase I part established the maximum Curr Probl Cancer, July/August 2008

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tolerated dose to be 10 mg orally once daily. Grade 3 toxicities included hyperglycemia (22%), hypophosphatemia (7%), fatigue (7%), diarrhea (4%), and anorexia (4%). Analysis of the phosphorylation of downstream targets of mTOR, eukaryotic initiation factor 4E-binding protein 1, and/or p70 S6 kinase showed inhibition in 66% of patients (6 of 9).44 Measuring S6K activity in peripheral blood mononuclear cells has been proposed as a surrogate of measuring the clinical effect of everolimus, given that prolonged inactivation of S6K1 has been noticed after intermittent treatment doses.120 When given as a single agent in mantle cell lymphoma cell lines, it induced G1 cell-cycle arrest and reduced phosphorylation of the mTOR downstream target, 4E-BP1. It also showed synergistic cytotoxicity with other agents that are commonly used in this disease, including doxorubicin, vincristine, or rituximab as well as other agents like bortezomib, paclitaxel, and vorinostat.121

Deforolimus (AP23573) A phase 2 clinical trial tested AP23573 in 12 patients with relapsed or refractory hematologic malignancies in which it was administered at a dose of 12.5 mg intravenously daily for 5 days every 2 weeks. The median age was 76 years. Three patients had minor responses in terms of hematologic improvement, and another 3 patients had stable disease. Serious drug-related side events were hypertriglyceridemia, mucositis, and neutropenic sepsis.122 A later report from the same study included 51 patients with a median age of 61 years. Serious side effects additionally reported were diarrhea, dyspnea, syncope, pleural effusion, and pneumonia. Nineteen of the 46 evaluable patients (41%) showed at least stable disease.123

Summary Despite significant advances in the treatment of hematological malignancies over the previous decades, morbidity and mortality from theses disorders still remain high. New discoveries in the pathways involved in the pathophysiology led to new thinking in drug discovery. mTOR inhibitors are new and viable options for patients with these malignancies. We hope that newer clinical data will bring about new options in this field.

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