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Dual Inhibition of Akt/Mammalian Target of Rapamycin Pathway by Nanoparticle Albumin-Bound-Rapamycin and Perifosine Induces Antitumor Activity in Multiple Myeloma

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Dual Inhibition of Akt/Mammalian Target of Rapamycin Pathway by Nanoparticle Albumin-Bound-Rapamycin and Perifosine Induces Antitumor Activity in Multiple Myeloma
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  Dual inhibition of Akt/mTOR pathway by nab  -rapamycin andperifosine induces anti-tumor activity in multiple myeloma Diana Cirstea 1 , Teru Hideshima 1 , Scott Rodig 2 , Loredana Santo 1 , Samantha Pozzi 3 , SoniaVallet 3 , Hiroshi Ikeda 1 , Giulia Perrone 1 , Kishan Patel 3 , Neil Desai 4 , Peter Sportelli 5 , ShwetaKapoor 6 , Shireen Vali 6 , Siddhartha Mukherjee 3 , Nikhil C. Munshi 1 , Kenneth C. Anderson 1 ,and Noopur Raje 1,3 1 Leebow Institute of Myeloma Therapeutics and Jerome Lipper Multiple Myeloma DiseaseCenter, Dana-Farber Cancer Institute 2 Department of Pathology, Brigham & Women’s Hospital 3 MGH Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston 4 Abraxis Bioscience LLC, Los Angeles 5 Keryx Biopharmaceuticals, Inc., New York 6 Cellworks Group Inc., Saratoga, CA Abstract The PI3K/Akt/mTOR pathway mediates multiple myeloma (MM) cell proliferation, survival, anddevelopment of drug resistance, underscoring the role of mTOR inhibitors such as rapamycin withpotential anti-MM activity. However, recent data demonstrate a positive feedback loop frommTOR/S6K1 to Akt, whereby Akt activation confers resistance to mTOR inhibitors. Weconfirmed that suppression of mTOR signaling in MM cells by rapamycin was associated withupregulation of Akt phosphorylation. We hypothesized that inhibiting this positive feedback by apotent Akt inhibitor perifosine would augment rapamycin-induced cytotoxicity in MM cells.Perifosine inhibited rapamycin-induced p-Akt, resulting in enhanced cytotoxicity in MM.1S cellseven in the presence of IL-6, IGF-1 or bone marrow stromal cells. Moreover, rapamycin inducedautophagy in MM.1S MM cells as evidenced by electron microscopy and immunocytochemistry,was augmented by perifosine. Combination therapy increased apoptosis detected by Annexin/PIanalysis and caspase/PARP cleavage. Importantly, in vivo  antitumor activity and prolongation of survival in a MM mouse xenograft model after treatment was enhanced with combination of nab -rapamycin and perifosine. Utilizing the in silico  predictive analysis we confirmed ourexperimental findings of this drug combination on PI3K, Akt, mTOR kinases, and the caspases.Our data suggests that mutual suppression of the PI3K/Akt/mTOR pathway by rapamycin andperifosine combination induces synergistic MM cell cytotoxicity, providing the rationale forclinical trials in patients with relapsed / refractory MM. Keywords myeloma; Akt; mTOR; apoptosis; autophagy Address correspondence to: Noopur Raje, MD, POB 216, MGH Cancer Center, Massachusetts General Hospital, 55 Fruit Street,Boston, MA 02114, nraje@partners.org, Phone: 617 726 0711, Fax: 617 724 6801. NIH Public Access Author Manuscript  Mol Cancer Ther  . Author manuscript; available in PMC 2011 May 17. Published in final edited form as: Mol Cancer Ther  . 2010 April ; 9(4): 963975. doi:10.1158/1535-7163.MCT-09-0763. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    INTRODUCTION Multiple myeloma (MM) is a bone marrow (BM) cancer driven by the interaction betweenclonal plasma cells and the BM microenvironment (1, 2). Among the major pathwaysmediating cytokine-induced MM cell growth and survival, PI3K/Akt/mTOR kinase cascadeplays a cardinal role in cell proliferation, survival and development of drug resistance (3-5).Cytokine-induced activation of Akt results in various down-stream anti-apoptotic effects viaBAD and forkhead transcription factor (FKHR) phosphorylation and inhibition of thecatalytic subunit of caspase-9. Besides its direct anti-apoptotic effects, p-Akt promotesgrowth and survival via phosphorylation of glycogen synthase kinase (GSK)-3 β  andmammalian target of rapamycin (mTOR). Moreover, Akt-induced activation of mTOR,enables mRNA translation through the activation of P70S6 kinase and the inhibition of 4E-BP1, a translational repressor of mRNAs. Consequently Akt which is constitutivelyactivated in MM patient cells and correlates with advanced stage and poor prognosis (6),represents a rational target for novel therapeutics.Identifying mTOR as a key kinase downstream of Akt led to the prediction that rapamycin, auniversal inhibitor of mTORC1-dependent S6K1 phosphorylation may be useful in thetreatment of MM (7-9).  In vitro  and in vivo  preclinical studies have demonstrated anti-MMactivity of rapamycin and its analogs (CCI-779 and RAD001) (10-14). First-generationmTOR inhibitors when used as single agents have demonstrated only modest efficacy inclinical trials (15-17), resulting in attempts to define mechanisms underlying rapamycinresistance. A growing body of evidence supports the hypothesis that resistance to rapamycinresults from a strong positive feedback loop from mTOR/S6K1 to Akt, resulting in Aktactivation (18-20). Indeed immunohistochemical analysis of paired tissue biopsies, beforeand after treatment with rapamycin-derivatives, revealed that non-responders frequentlydevelop increased p-Akt, supporting the view that increased intra-tumoral phosphorylationof Akt mediates rapamycin resistance (21, 22).The low response rate observed in many tumor types to rapamycin-derivatives led to twostrategies to overcome rapamycin resistance. First, the implementation of nano-particlealbumin-bound ( nab ) technology to augment rapamycin delivery to tumor tissue (23, 24).Second, combination strategies such as rapamycin with lenalidomide with the ability toovercome the protective effects of growth factors in the tumor milieu are in use (10).Given that mTOR inhibitors induce PI3K/Akt activity in MM cells (25), we have examinedthe utility of adding an Akt inhibitor to overcome mTOR resistance and have also taken theadvantage of nano-particle technology with nab -rapamycin. To date, the best-characterizedand most developed clinical inhibitor of Akt is the novel alkylphospholipid, perifosine (26,27). We first confirmed that suppression of mTOR signaling by rapamycin was associatedwith upregulation of Akt activation. We therefore inquired whether perifosine could: (i)inhibit rapamycin-induced p-Akt; (ii) augment rapamycin-induced cytotoxicity in vitro ; and(iii) translate into enhanced in vivo  anti-tumor activity when used with the nab -basedrapamycin (ABI-009). Our data suggests that rapamycin-induced cytotoxicity waspredominantly triggered as a consequence of autophagy in MM cells. The combination of rapamycin and perifosine resulted in 2 cell death-inducing events: autophagy and apoptosis.Furthermore, the combination of nab -rapamycin and perifosine resulted in significantantitumor activity in an in vivo  human MM cell xenograft murine model. Finally, utilizingthe in silico  predictive analysis based on a systems biology approach (28, 29) we confirmedour experimental findings regarding the biological effects of this drug combination. Thesestudies therefore provide the preclinical rationale for combination clinical trials in patientswith MM. Cirstea et al.Page 2  Mol Cancer Ther  . Author manuscript; available in PMC 2011 May 17. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    MATERIALS AND METHODS Cell culture and reagentsMM derived cell lines— Dexamethasone (Dex) –sensitive (MM.1S) MM cell line wasprovided by Dr. Steven Rosen (Northwestern University, Chicago, IL). The INA-6 cell linewas kindly provided by Dr Martin Gramatzki (University of Erlangen-Nuernberg, Erlangen,Germany). OPM1 cell line was provided by Dr P. Leif Bergsagel (Mayo Clinic, Scottsdale,AZ). All MM cell lines were cultured in RPMI-1640 containing 10% fetal bovine serum(FBS; Sigma Chemical, St Louis, MO), 2 M L-glutamine, 100 U/mL penicillin, and 100 g/ mL streptomycin (GIBCO, Grand Island, NY). Generation of bone marrow stroma cells(BMSCs) from BM specimens from MM patients obtained after appropriate IRB approvedinformed consent has been previously described (10). Once confluent, the cells weretrypsinized and passaged as needed. BMSC were incubated in 96 well culture plates(approximately 5000 to 10,000 BMSC/well) for 24 hours, MM.1S cells were then added tothe wells (2 × 10,000 cells/well) and incubated with media alone, rapamycin, perifosine, orcombination for 48 hours at 37°C at the specified concentrations. Rapamycin— Rapamycin was obtained from Calbiochem (San Diego, CA). Perifosine— Perifosine (NSC 639966), a synthetic substituted heterocyclicalkylphospholipid, was provided by Keryx Biopharmaceuticals (New York, NY). nab  -rapamycin— nab -rapamycin (ABI-009) was provided by Abraxis Bioscience LLC(Los Angeles, CA). Akti-½— Akti-½ was procured from Calbiochem (San Diego, CA). Cell viability and proliferation assaysColorimetric assay— Colorimetric assays were performed to assay drug activity. Fortyeight hour cultures were pulsed with 10 μ L of 5 mg/mL 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrasodium bromide (MTT; Chemicon International Inc, Temecula, CA) to eachwell, followed by incubation at 37°C for 4 hours, and addition of 100 μ L isopropanol with0.04 HCl. Absorbance readings at a wavelength of 570 nm (with correction using readings at630 nm) were taken on a spectrophotometer (Molecular Devices Corp., Sunnyvale, CA). Proliferation assay— DNA synthesis was measured by tritiated thymidine uptake [3H-TdR] (Perkin Elmer, Boston, MA) as previously described (10). Briefly, MM.1S cells (2-3 ×10.000 cells/well) were incubated in 96-well culture plates alone or in co-culture withBMSCs, recombinant IL-6 (10 ng/mL) or IGF-1 (50 ng/mL) in the presence of media orvarying concentrations of rapamycin, perifosine, or combination for 48 hours at 37°C. Immunoblotting MM cells were harvested and whole-cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane(Bio-Rad Laboratories, Hercules, CA), as described previously (10). The antibodies used forimmunoblotting included: anti–phospho (p)-Akt (Ser473), anti-Akt, anti–phospho (p)-P70S6K, anti-P70S6K, anti-GAPDH, anti–caspase-8, anti–caspase-3, anti-caspase-9, anti-PARP, and anti-tubulin (Cell Signaling Technology Inc., Beverly, MA, USA). Cirstea et al.Page 3  Mol Cancer Ther  . Author manuscript; available in PMC 2011 May 17. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    Detection of apoptosis by Annexin V/PI staining Detection of early apoptotic cells was performed with the annexin V-PI detection kit(Immunotech/Beckman Coulter). Briefly, 10 6  MM cells were exposed for 24-48 hours torapamycin (10 nM), perifosine (5 uM), or combination, washed and then incubated in thedark at room temperature with annexin V-FITC and PI for 15 minutes. Annexin V + PI - apoptotic cells were enumerated using the Epics flow cytometer. Cells that were annexin V-FITC1 positive (with translocation of phosphatidylserine from the inner to the outer leafletof the plasma membrane) and PI negative (with intact cellular membrane) were consideredas early apoptotic cells, while positivity for both annexin V-FITC1 and PI was associatedwith late apoptosis or necrosis. Immunocytochemical detection of LC3 MM.1S cells were cultured in the presence of media, 10 nM rapamycin, 5 uM perifosine, orcombination for 3 hours at 37°C, and cytospins were prepared. Cells were fixed in 4%paraformaldehyde. The anti-LC3 polyclonal antibody (MBL I.C.) was diluted with PBS at1:100 and incubated with cells overnight at 4°C. FITC-conjugated anti-rabbit IgG at 1:100dilutions was added for 1 hour at 4°C, then DAPI containing mounting medium and coverslips added promptly. Samples were observed by fluorescence microscopy and digitallyphotographed. Electron microscopy MM.1S cells were cultured in the presence of media or 10 nM rapamycin, 5 uM perifosine,or combination for 3 and 16 hours at 37°C. Cells were collected and fixed with 2.0%paraformaldehyde/2.5% EM grade glutaraldehyde in 0.1 M sodium cacodylate buffer (pH7.4) at 37°C. After fixation, samples were placed in 2% osmium tetroxide in 0.1 M sodiumcacodylate buffer (pH 7.4), dehydrated in a graded series of ethyl alcohol, and embedded inresin. Ultrathin sections were cut and placed on formvar-coated slot copper grids. Sectionswere then counterstained with uranyl acetate and lead citrate, and viewed with a TecnaiTMG2 Spirit Bio TWIN electron microscope. Digital images were acquired with an AMT 2k CCD camera (direct magnification 1400X and 6800X). In silico   study In silico study was performed using the iC-PHYS™ Oncology Technology (CellworksGroup Inc (CWG), India) (30, 31). The iC-PHYS™ Oncology platform consists of adynamic representation of the signaling pathways underlying tumor physiology at the bio-molecular level. All the key relevant proteins and associated gene and mRNA transcriptswith regard to tumor related signaling are comprehensively included in the system with theirrelationship quantitatively represented. The modeling of time dependent changes in thefluxes of the constituent pathways has been carried out utilizing modified OrdinaryDifferential Equations (ODE) and Mass Action Kinetics. The state of the system was set tosimulate late tumor stage. The drug concentrations used in the model is assumed to be postADME (Absorption, Distribution, Metabolism, Excretion). The bottom layer is thecomputational backplane which enables the system to be dynamic and computes all themathematics in the middle layer. The Oncology platform is ported to iC-PHYS™ and issimulated so that all the molecules attain the control steady state values (~by 1 × 10 5 seconds), following which the triggers are introduced in to the system. This leads to a phaseof disease progression and the model stabilizes at steady disease levels by 2 × 10 5  seconds.In initial conditions, the model simulated the kinetic interactions of the PI3K/Akt/mTorinteractome based on proteomic data characterizing the pathophysiology of late stage cancerdisease. Rapamycin: 10 nM; Ki: 1e-2, perifosine: 5 uM; Ki: 3.79e-1 uM, and their Cirstea et al.Page 4  Mol Cancer Ther  . Author manuscript; available in PMC 2011 May 17. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t    combination were tested on the system to observe the consequent effects on mTOR, p-Akt,and caspases levels. MM xenograft murine model The in vivo  anti-MM activity of both single agent nab -rapamycin, perifosine, and thecombination of nab- rapamycin and perifosine treatment was evaluated in CB-17 severecombined immunodeficient (SCID) mice obtained from Charles River Laboratories(Wilmington, MA). Housed and monitored in the Animal Research Facility at the Dana-Farber Cancer Institute (DFCI), mice were subjected to animal studies according to theprotocols approved by the Animal Ethics Committee. Forty male 5-6 week old mice wereirradiated (2 Gy [200 rad]) using cesium 137 ( 137 Cs) -irradiator source); 24 hours afterirradiation 2.5 × 10 6  MM.1S cells suspended in 100 μ L of RPMI medium were inoculatedsubcutaneously. When tumors were measurable, mice were randomly assigned into cohorts(10 mice per cohort) receiving nab -rapamycin (10 mg/kg by tail vein injections on days 1, 3,5 weekly for 4 weeks), perifosine (125 mg/kg orally by gavage on day 5 weekly for 4weeks), or both ( nab -rapamycin on day 1, 3, 5 with perifosine added at day 5 weekly, for 4weeks). Control mice (n=10) were administered vehicles: PBS orally and 0.9% sodiumchloride by tail vein on the same schedule as the combination. Animals were monitored forbody weight and tumor volume by caliper measurements every alternate day. Tumor volumewas estimated using the following formula: ½ × (length) × (width) 2 . Animals wereeuthanized in accordance with institutional guidelines by CO 2  inhalation in the event of tumor size > 2cm or major compromise in their quality of life, due to tumor ulceration.Survival was evaluated from the first day of treatment until death. Tumor growth wasevaluated using caliper measurements from the first day of treatment until day of firstsacrifice, which was day 33 for controls, day 47 for nab -rapamycin-treated, day 47 forperifosine-treated and day 89 for combination-treated cohorts. Immunohistochemical staining Immunohistochemical staining was performed using the standard avidin-biotin complex-peroxidase method on formalin-fixed, paraffin embedded tissue sections of tumor excisedfrom xenografts following one week treatment with either nab -rapamycin (10mg/kg by tailvein thrice weekly), perifosine (125 mg/kg orally by gavage once a week), both, or controlvehicles. Tumor sections were stained with anti–phospho (p)-Akt (Ser473), or cleavedcaspase-3 antibody (Cell Signaling Technology Inc.), or LC3 antibody APG8a N-term(Abgent, Inc., San Diego, CA, USA), or were subjected to TUNEL staining. Statistical analysis All in vitro  experiments were performed in triplicate and repeated at least 3 times; arepresentative experiment was selected for figures. Statistical significances of differenceswere determined using Student t test, with minimal level of significance P  < 0.05. Allstatistical analysis of the in vivo  data was determined using GraphPad prism software(GraphPad Software, Inc. San Diego, CA, USA). Synergism was determined by using theChou-Talalay method (32). RESULTS Rapamycin induces p-Akt in MM cells, while perifosine inhibits p-Akt To confirm the effects of rapamycin signaling on MM cells, MM.1S cells were exposed toincreasing concentrations of rapamycin for 2 hours. Rapamycin treatment resulted in a dose-dependent decrease of p-P70S6K. This was accompanied by an increase in phosphorylationof Akt at Ser473, starting at doses as low as 1 nM. Inhibition of p-P70S6K and activation of  Cirstea et al.Page 5  Mol Cancer Ther  . Author manuscript; available in PMC 2011 May 17. N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  N I  H -P A A  u t  h  or M an u s  c r i   p t  
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