`
`Current Cancer Drug Targets, 2010, 10, 155-167
`
`155
`
`to Inhibit Multiple
`Dexamethasone Synergizes with Lenalidomide
`Myeloma Tumor Growth, But Reduces Lenalidomide-Induced Immuno-
`modulation of T and NK Cell Function
`
`A.K. Gandhi*,1, J. Kang1, L. Capone1, A. Parton1, L. Wu1, L.H. Zhang1, D. Mendy2, A. Lopez-
`Girona2, T. Tran2, L. Sapinoso2, W. Fang2, S. Xu2, G. Hampton2, J.B. Bartlett1 and P. Schafer1
`
`1Celgene Corporation, Summit, New Jersey, USA, 2Celgene Corporation, San Diego, California, USA
`
`Abstract: To determine the effect of dexamethasone on the antimyeloma effects of lenalidomide, we tested in vitro pro-
`liferation, tumor suppressor gene expression, caspase activity, cell cycling, and apoptosis levels in a series of multiple
`myeloma (MM) and plasma cell leukemia cell lines treated with lenalidomide and dexamethasone, alone or in combina-
`tion. The effect of dexamethasone on the immunomodulatory activities of lenalidomide such as T cell and natural killer
`(NK) cell activation was measured via interleukin [IL]-2 production, and interferon-(cid:1) and granzyme B production respec-
`tively. Lenalidomide inhibited proliferation in most cell lines tested, and this effect was enhanced by dexamethasone. This
`effect was observed in MM cells containing the high-risk cytogenetic abnormalities t(4;14), t(14;16), del17p, del13, and
`hypodiploidy. Mechanistically, lenalidomide plus dexamethasone synergistically induced expression of the tumor sup-
`pressor genes Egr1, Egr2, Egr3, p15, p21, and p27 in MM cell lines and MM patient cells. The combination activated
`caspases 3, 8, and 9 and induced cell cycle arrest and apoptosis. Lenalidomide alone increased T cell production of IL-2,
`and NK cell production of interferon-(cid:1) and granzyme B. Notably, dexamethasone antagonized these immunostimulatory
`effects of lenalidomide in a dose-dependent manner. These data further elucidate the mechanism of action of lenalidomide
`and dexamethasone in MM, and suggest that use of low-dose dexamethasone with lenalidomide may retain the antiprolif-
`erative effect of lenalidomide while permitting greater immunomodulatory effects of this combination regimen.
`
`Keywords: Myeloma, lenalidomide, dexamethasone, proliferation, immunomodulation.
`
`INTRODUCTION
`
`Lenalidomide is an immunomodulatory agent that is ap-
`proved for use in combination with dexamethasone in pa-
`tients with multiple myeloma (MM) who have received at
`least one prior therapy. Recently reported evidence from a
`randomized trial conducted in patients with newly diagnosed
`MM suggests that overall survival was prolonged when dex-
`amethasone was given less frequently (low-dose dexametha-
`sone regimen defined as 40 mg/day on days 1, 8, 15, and 22
`of each 28-day cycle) in combination with standard le-
`nalidomide, compared with standard dexamethasone therapy
`(40 mg/day on days 1–4, 9–12, and 17–20 of each 28-day
`cycle) in combination with standard lenalidomide.1
`
`Lenalidomide has multiple effects on myeloma cells and
`their microenvironment which may contribute to its antican-
`cer effects [1], including direct antiproliferative effects on
`myeloma cells [2], increasing T cell cytokine production [3],
`and immunomodulatory effects via natural killer (NK) cell
`activity [4]. The mechanism of direct myeloma tumor
`growth inhibition by lenalidomide has been shown, in some
`cases, to involve upregulation of the tumor suppressor gene
`
`
`
`
`*Address correspondence to this author at Drug Discovery, Celgene Corpo-
`ration, 86 Morris Avenue, Summit, New Jersey 07901, USA; Tel: (908) 673
`9633; Fax: (908) 673 2788; E-mail: agandhi@celgene.com
`
`
`1Rajkumar, S. V.; Jacobus, S.; Callander, N.; Fonseca, R.; Vesole, D.; Williams, M. V.;
`Abonour, R.; Siegel, D. S.; Katz, M.; Greipp, P. R. Randomized trial of lenalidomide
`plus high-dose dexamethasone versus lenalidomide plus low-dose dexamethasone in
`newly diagnosed myeloma (E4A03), a trial coordinated by the Eastern Cooperative
`Oncology Group: Analysis of response, survival, and outcome. J. Clin. Oncol. 2008,
`26(Suppl), abstract 8504.
`
`
`
`1568-0096/10 $55.00+.00
`
`p21 [1, 2], and activation of caspases [5, 6]. Although the
`antiproliferative effects of lenalidomide play a key role in
`the antimyeloma mechanism, the immunomodulatory effects
`of lenalidomide also contribute to its anticancer activity.
`Lenalidomide co-stimulates T cells to proliferate, produce
`several cytokines including interleukin (IL)-2, IL-5, IL-10
`and interferon (IFN)-(cid:1), and increase expression of CD40
`ligand [3, 7-9]. The immunomodulatory effects of lenalido-
`mide also involve NK cell activity and antibody-dependent
`cellular cytotoxicity. In purified human NK cells, lenalido-
`mide enhances the production of IFN-(cid:1) and granzyme B [4],
`as well as enhancing NK cell-mediated lysis of both MM cell
`lines and MM patient cells [10].
`
`As with other glucocorticoids, dexamethasone targets
`steroid receptors and affects many biological processes in-
`cluding glucose and protein metabolism, and inflammation.
`Dexamethasone is a potent anti-inflammatory agent, which is
`used in various therapeutic areas, including dermatology,
`hematology, and endocrinology. The anti-inflammatory ef-
`fects of dexamethasone are produced via several mecha-
`nisms, including inhibition of phospholipase A2, release of
`arachidonic acid from cellular lipids, and inhibition of pros-
`taglandin H2 synthase or cyclooxygenase-2 expression [11].
`Dexamethasone’s anti-proliferative activity involves upregu-
`lation of p21 protein expression and inhibition of CDK2 ac-
`tivity and retinoblastoma (Rb) phosphorylation [12]. The
`anti-inflammatory and antiproliferative properties of dex-
`amethasone have made it part of the conventional therapy
`regimen for MM along with alkylating agents and anthracy-
`clines.
`
`© 2010 Bentham Science Publishers Ltd.
`
`ALVOGEN, Exh. 1045, p. 0001
`
`

`

`156 Current Cancer Drug Targets, 2010, Vol. 10, No. 2
`
`Gandhi et al.
`
`Although the individual effects of lenalidomide and dex-
`amethasone on proliferation and immunomodulation have
`been studied, the combination effects of lenalidomide to-
`gether with dexamethasone have not been thoroughly ex-
`plored, nor has the mechanism of this combination been ex-
`amined. Therefore, we conducted a series of experiments in
`various myeloma cell lines to determine the effects of dex-
`amethasone on the antiproliferative, apoptotic and immuno-
`modulatory activity of lenalidomide. In addition, tumor sup-
`pressor gene expression and caspase activity were assessed
`in MM cell lines and MM patient cells to identify any syner-
`gistic effects of combining lenalidomide with dexametha-
`sone.
`
`MATERIALS AND METHODS
`
`Multiple Myeloma and Plasma Cell Leukemia Cell Lines
`
`Human cell lines EJM, L363, KMS-12-BM, KMS-12-
`PE, Karpas-620, SKMM2, JJN-3, OPM-2, and LP-1 (DSMZ,
`Braunschweig, Germany), and cell lines RPMI-8226 (le-
`nalidomide-resistant), U266 B1, and NCI-H929 (ATCC,
`Manassas, VA) were obtained.
`
`Cell Proliferation Assay
`Cell proliferation was assessed by the 3H-thymidine in-
`corporation assay. Briefly, cells were cultured in 96-well cell
`culture plates in the presence or absence of lenalidomide,
`dexamethasone, or both. Each well contained 6000 cells/80
`μL cell culture medium (Roswell Park Memorial Institute
`(RPMI)-1640 + 10–20% fetal bovine serum (FBS), 1%
`pen/strep/1% L-glutamine). Compound dilutions were made
`in 10(cid:2) the required final concentration, and 10 μL of each
`compound was added to the cells in triplicate. The cells
`were treated with lenalidomide at 0.0001–100 μM final, or
`with dexamethasone at 0.000098–0.4 μM (Calbiochem, San
`Diego, CA), in a final concentration of 0.2% dimethyl sul-
`foxide (DMSO) for all samples. Cells were grown at 37°C in
`a humidified incubator at 5% CO2 for 72 hours in the pres-
`ence of the test compounds. One microcurie of 3H-thymidine
`(GE Healthcare, Fairfield, CT) was added to each well for
`the final 6 hours of culture. The cells were harvested onto
`UniFilter-96 GF/C filter plates (PerkinElmer, Waltham,
`MA) using a cell harvester (Tomtec, Hamden, CT), and the
`plates were allowed to dry overnight. A total of 25 μL/well
`of Microscint™-20 (PerkinElmer) was added and the plates
`were analyzed in TopCount NXT (PerkinElmer). Each well
`was counted for 1 minute. The percentage inhibition of cell
`proliferation was calculated by averaging all triplicates and
`normalizing to the DMSO control (0% inhibition). Final cu-
`mulative half-maximal inhibitory concentrations (IC50) were
`calculated using non-linear regression and sigmoidal dose-
`response, constraining the top to 100% and bottom to 0%
`and allowing variable slope, using GraphPad Prism version
`5.01. SEM (standard error of the mean) was calculated from
`the individual IC50s of each replicate.
`
`ATP Production Assay
`
`For each cell line, 40 μL of cells were seeded in each
`well of a 384-well flat, clear bottom, black polystyrene, TC-
`Treated plate (Corning Inc., Life Sciences, Lowell, MA) at
`
`an optimized density to ensure that the cell growth was
`within the linear detection range after 4 days in culture. For
`single-agent treatment, lenalidomide or dexamethasone was
`serially diluted 3-fold for 9 concentrations and added to the
`cells with the final DMSO concentration of 0.2%. The
`maximum concentrations for lenalidomide and dexametha-
`sone were 30 μM and 1 μM, respectively. For combination,
`lenalidomide and dexamethasone were titrated orthogonally
`in 3-fold dilution for 9 series and added to the cells. The
`384-well plate was then sealed with Breathe-Easy™ sealing
`membrane (Sigma-Aldrich, St. Louis, MO) and incubated at
`37°C. After 96 hours, the cell viability was determined using
`CellTiter-Glo® Luminescent Cell Viability Assay (Promega
`Corp., Madison, WI) following the manufacturer’s instruc-
`tions. Background subtracted luminescence counts were
`converted to percentages of cell viability with respect to
`DMSO treated control cells. Dose response curves were gen-
`erated using XLfit4 (IDBS, Surrey, UK) by fitting the per-
`centage of control data at each concentration using a four-
`parameter logistic model/sigmoidal dose-response model: (y
`= (A+((B-A)/(1+((C/x)^D))))); where A = minimum, B =
`maximum, C = IC50, and D = Hill slope. Average half-
`maximal inhibitory concentration (IC50) and SEM (standard
`error of the mean) was calculated from the individual IC50s of
`each replicate.
`
`Gene Expression Analysis
`Myeloma cell lines (1 (cid:2) 106 cells/sample) were cultured
`in 2 mL RPMI-1640 medium + 10% FBS with 0.2% DMSO,
`and lenalidomide (1 or 10 μM) with or without dexametha-
`sone (800 nM) for 6 hours and 24 hours. Ficoll-purified pe-
`ripheral blood mononuclear cells from a newly diagnosed
`MM patient (Proteogenex, Culver City, CA) were thawed
`and resuspended in 40% original freezing medium, 40%
`conditioned media, 20% autologous donor serum (Pro-
`teoGenex) and 1 ng/mL recombinant IL-6 (R&D Systems,
`Minneapolis, MN). Patient cells were seeded in a 48-well
`plate at 1 (cid:2) 106 cell in 0.5 mL and treated with 0.2% DMSO,
`10 μM lenalidomide, 800 nM dexamethasone or 10 μM le-
`nalidomide/800 nM dexamethasone for 24 hours. Condi-
`tioned media was prepared by co-culturing 5 (cid:2) 105 HS-5
`human bone marrow stromal cells (ATCC) with 1 (cid:2) 105 NCI-
`H929 MM cells (ATCC) in 4 mL RPMI-1640 media with
`10% premium FBS for 24 hours and media was spun to re-
`move cells. After compound treatment, cells were harvested
`and total RNA was purified with RNAeasy (Qiagen, Valen-
`cia, CA). Quantitative reverse transcriptase polymerase chain
`reaction (qRT-PCR) was performed. Gene expression assays
`were performed using a 7500 Real-Time PCR System (Ap-
`plied Biosystems, Foster City, CA) and analyzed using the
`relative quantification method for the early growth response
`(Egr) genes Egr1, Egr2 and Egr3, and the cyclin-dependent
`kinase (CDK) inhibitors p15, p16, p21, and p27. For each
`sample, a GAPDH control was used for normalization. For
`each gene, samples within each experiment were normalized
`to a control (0.2% DMSO only) for each time point and cell
`line. Gene expression results are expressed as 2-ddCt.
`
`Western Blot Analysis
`Protein extracts made from 5 x 106 cells were subjected
`to 4–12% Bis-Tris Criterion XT gel (Bio-Rad Laboratories,
`Hercules, CA) and transferred onto a 0.45 (cid:1)M nitrocellulose
`
`ALVOGEN, Exh. 1045, p. 0002
`
`

`

`Dexamethasone Synergizes with Lenalidomide
`
`Current Cancer Drug Targets, 2010, Vol. 10, No. 2 157
`
`criterion membrane. Membranes were probed with primary
`rabbit phospho-antibodies pp-RbSer807/811, pp-RbSer608
`and anti-p21 and Egr1 antibodies (Cell Signaling Technol-
`ogy Inc., Danvers, MA), mouse anti-p27 antibody (BD Bio-
`sciences, San Jose, CA) and actin antibody (Sigma-Aldrich)
`followed by Alexa-conjugated secondary antibody (Invitro-
`gen Corporation, Carlsbad, CA). Immunoreactive bands
`were visualized and quantified with Odyssey Infrared Imag-
`ing (LI-COR Biosciences, Lincoln, NE).
`
`Caspase Assay
`Myeloma cells (0.5 (cid:1) 106–1 (cid:1) 106 cells/sample) were cul-
`tured in 4 mL RPMI-1640 + 10–20% FBS with 0.2%
`DMSO. Caspases 3, 8, and 9 fluorescent-based assays (R&D
`Systems) were performed after 24 and 48 hours in cells from
`various myeloma cell lines incubated with lenalidomide (1
`or 10 μM) with or without dexamethasone (1 μM).
`Staurosporine (10 μM; Alexis Biochemicals, Plymouth
`Meeting, PA) was used as a positive control for caspase ac-
`tivation; positive control data were collected after 24 hours
`only. Cell lysates were made and all samples were normal-
`ized to protein concentration using the BCA Protein Assay
`(Thermo Scientific, Rockford, IL). Caspase activity was
`measured by fluorescence wavelengths 420/50 nm (excita-
`tion) and 530/25 nm (emission) (FLx800 Fluorescence Mi-
`croplate Reader; BioTek Instruments Inc., Winooski, VT).
`
`Cell Cycle Analysis
`
`Myeloma cell lines were treated with lenalidomide (1 or
`10 μM), dexamethasone (0.8 or 1 μM), or both for 72 hours;
`lenalidomide was added once daily. Cell cycle profiling was
`performed using BD Cycletest™ Plus (BD Biosciences) and
`analyzed on a FACSCanto™ flow cytometer (BD Bio-
`sciences).
`
`Apoptosis Analysis
`
`Cell lines LP-1 (lenalidomide-sensitive) and RPMI-8226
`(lenalidomide-resistant) were treated with lenalidomide (1 or
`10 μM), dexamethasone (1 μM), or both for 72 hours. Apop-
`totic cells were stained with propidium iodide and annexin
`V, and analyzed using a FACSCanto™ flow cytometer (BD
`Biosciences).
`
`T Cell IL-2 Production
`
`Human T cells were isolated from leukocyte units by
`negative selection using CD16, CD33, CD56, CD14, CD19,
`and human leukocyte antigen class II antibodies (Invitrogen).
`The T cells were plated at 2.5 (cid:1) 105 cells/well in 96-well
`plates pre-coated with anti-CD3 monoclonal antibody (2.5
`μg/mL). Lenalidomide (0.0016–10 μM) was added alone or
`in combination with dexamethasone (0.3–10 nM). The
`plates were incubated for 48 hours at 37°C. Supernatants
`from two donors were then harvested and analyzed in the
`human IL-2 enzyme-linked immunosorbent assay (R&D
`Systems).
`
`NK Cell IFN-(cid:1) Production
`
`NK cells were isolated from leukocyte units by a 30-
`minute incubation with RosetteSep® cocktail (StemCell
`Technologies, Inc., Vancouver, BC, Canada) followed by
`Ficoll-Hypaque density gradient centrifugation. The CD56+
`NK cells were isolated to approximately 85% purity as de-
`
`termined by flow cytometry. Flat-bottom plates were coated
`with 100 μg/mL of human immunoglobulin (Ig)G (Sigma-
`Aldrich) overnight at 4°C, and unbound IgG was removed.
`The NK cells were plated at 2 (cid:1) 105 cells/well in 96-well
`plates, and 10 ng/mL of IL-2 (R&D Systems) and 0–10 μM
`lenalidomide were added. After 48 hours, supernatants were
`harvested and analyzed for levels of IFN-(cid:1) (R&D Systems)
`and granzyme B (Cell Sciences, Inc., Canton, MA).
`
`Calculation of Synergy/Additive Effects
`
`To evaluate the combinatory effect of lenalidomide and
`dexamethasone on MM cell lines, data from the two inde-
`pendent experiments were analyzed by comparing the com-
`binatory response against the theoretical additive response of
`the two agents. The expected additive effect of two agents
`(A and B) was calculated using the fractional product
`method [13]: (fu)A,B = (fu)A (cid:1) (fu)B; where fu = fraction
`unaffected by treatment.
`
`A synergism of a combination is determined when the
`observed fraction unaffected in combination is less than
`(fu)A,B, whereas an additive effect is determined when the
`observed fraction unaffected in combination equals (fu)A,B.
`
`RESULTS
`
`Antiproliferative Activity
`
`Lenalidomide and Dexamethasone Inhibit the Growth of
`MM Cells
`
`The effects of lenalidomide alone, dexamethasone alone,
`and lenalidomide plus dexamethasone on the growth of MM
`cells were evaluated with a 72-hour 3H-thymidine incorpora-
`tion assay. The growth inhibition effects of lenalidomide and
`dexamethasone as single agents were determined in 10 MM
`cell lines. As shown in Table 1, lenalidomide alone was most
`potent in the Karpas-620, KMS-12-BM, and NCI-H929
`cells. Dexamethasone alone induced inhibition at sub-
`micromolar concentrations in most MM cells tested. The
`antiproliferative effects of lenalidomide plus dexamethasone
`ranged from non-additive (Karpas-620, KMS-12-BM, and
`EJM) to partially-additive (NCI-H929, U266 B1, JJN-3,
`SKMM2, and RPMI-8226) and additive (LP-1 and OPM-2).
`Non-additive effects indicate that the combination has no
`greater effect than either lenalidomide or dexamethasone
`alone, whichever was greater.
`
`As no synergies were detected by the thymidine incorpo-
`ration assay, an ATP production assay was performed to
`assess all viable cells after a longer, 96 hour, drug treatment
`period. The ATP assay is a less sensitive assay because it
`measures all viable cells, including arrested cells, whereas
`the thymidine incorporation assay measures only those cells
`undergoing active DNA synthesis. The growth inhibition
`effects of lenalidomide and dexamethasone as single agents
`were determined in seven MM cell lines. Lenalidomide
`alone was most potent in the KMS-12-BM and NCI-H929
`cells (Table 2). Dexamethasone alone induced inhibition at
`sub-micromolar concentrations in OPM-2, NCI-H929, and
`SKMM2 cells. Although the individual inhibitory concentra-
`tions were higher in the ATP assay compared with the
`thymidine
`incorporation assay,
`lenalidomide plus dex-
`amethasone produced synergistic effects on ATP inhibition
`
`ALVOGEN, Exh. 1045, p. 0003
`
`

`

`158 Current Cancer Drug Targets, 2010, Vol. 10, No. 2
`
`Gandhi et al.
`
`Table 1.
`
`Inhibition of Proliferation in Myeloma Cell Lines Treated with Lenalidomide and Dexamethasone for 72 Hours, as
`Measured by the 3H-Thymidine Incorporation Assay (n=3)
`
`
`
`
`
`Cell Line
`
`Karpas-620
`
`KMS-12-BM
`
`NCI-H929
`
`LP-1
`
`U266 B1
`
`JJN-3
`
`SKMM2
`
`OPM-2
`
`EJM
`
`RPMI-8226
`
`Lenalidomide
`
`IC50 in μM (SEM)
`
`0.046 (0.005)
`
`0.057 (0.01)
`
`0.08 (0.03)
`
`2.1 (1.1)
`
`3.7 (0.9)
`
`9.9 (6.0)
`
`18 (4.0)
`
`32 (17)
`
`99 (64)
`
`>100
`
`Dexamethasone
`
`IC50 in μM (SEM)
`
`0.041 (0.02)
`
`0.051 (0.05)
`
`0.0043 (0.001)
`
`0.018 (0.005)
`
`0.19 (0.05)
`
`0.061 (0.02)
`
`0.0077 (0.0008)
`
`0.02 (0.0054)
`
`> 1.0
`
`Combination Effect
`
`Non-additive
`
`Non-additive
`
`Partially additive
`
`Additive
`
`Partially additive
`
`Partially additive
`
`Partially additive
`
`Additive
`
`Non-additive
`
`0.0054 (0.0019)
`
`Partially additive
`
`Table 2.
`
`Inhibition of Myeloma Cell Lines Treated with Lenalidomide and Dexamethasone for 96 Hours, as Measured by the ATP
`Production Assay (n=2)
`
`
`
`
`
`Cell Line
`
`LP-1
`
`OPM-2
`
`NCI-H929
`
`KMS-12-PE
`
`U266 B1
`
`L363
`
`SKMM2
`
`Lenalidomide IC50
`in μM (SEM)
`
`Dexamethasone IC50 in
`μM (SEM)
`
`Effect
`
`High Risk Cytogenetic Feature
`
`> 30
`
`3 (1.0)
`
`1.78 (0.9)
`
`0.74 (n=1)
`
`> 30
`
`> 30
`
`> 30
`
`> 1
`
`0.069 (0.012)
`
`0.04 (0.008)
`
`> 1
`
`> 1
`
`> 1
`
`0.012 (0.0006)
`
`Synergy
`
`Synergy
`
`Synergy
`
`Synergy
`
`Non-additive
`
`Non-additive
`
`Non-additive
`
`del13
`
`t(4;14), hypodiploid
`
`del13, hypodiploid
`
`del(13)(q11), 4(11;14), hypodiploid
`
`del13, hypodiploid
`
`del(17p)(p12)
`
`del13, hypodiploid
`
`in the LP-1, OPM-2, NCI-H929, and KMS-12-PE cells. The
`discrepancy between the combination effects observed with
`the two assays can be attributed to the longer duration of
`drug treatment in the ATP assay. LP-1 cells treated with
`constant lenalidomide and titrated dexamethasone (Fig. 1A)
`or constant dexamethasone and titrated lenalidomide (Fig.
`1B) synergistically inhibited cell viability. Similar results
`were seen in OPM-2 cells, where treatment with constant
`lenalidomide and titrated dexamethasone (Fig. 1C) or con-
`stant dexamethasone and titrated lenalidomide (Fig. 1D)
`synergistically inhibited cell viability.
`
`Lenalidomide and Dexamethasone Enhance Expression of
`Tumor Suppressor Genes
`
`Upregulation of the tumor suppressor gene p21 in MM
`has been described as a mechanism of action of both dex-
`amethasone [1, 12] and lenalidomide [2]. The effects of le-
`nalidomide and dexamethasone, alone or in combination, on
`tumor suppressor gene expression were evaluated by qRT-
`PCR in 6 MM cell lines and in cells taken from a MM pa-
`tient. The results from all cell lines are summarized in Table
`3 and show that lenalidomide and dexamethasone alone can
`enhance expression of tumor suppressor genes p15, p21,
`Egr1, Egr2, and Egr3 differentially in different cell lines.
`The combination of lenalidomide and dexamethasone syner-
`
`gistically induced gene expression of p15 in Karpas-620
`cells, p21 in NCI-H929, LP-1, U266 B1, JJN-3 and RPMI-
`8226, and p27 expression in NCI-H929 and RPMI-8226.
`Lenalidomide and dexamethasone also synergistically en-
`hanced Egr1 in Karpas-620 and JJN-3 cells, Egr2 in JJN-3
`and RPMI-8226, and Egr3 in Karpas-620, NCI-H929 and
`JJN-3. Gene expression of Egr1, Egr2, and Egr3 were syn-
`ergistically induced by the combination in MM patient cells.
`After 24 hours, LP-1 cells treated with lenalidomide or dex-
`amethasone as single agents enhanced p21 expression 9-fold
`and 12-fold, respectively, whereas the combination enhanced
`p21 expression up to 65-fold (Fig. 2A). JJN-3 cells treated
`with both lenalidomide and dexamethasone synergistically
`enhanced expression of Egr1, Egr2, Egr3, and p21 (Fig. 2B).
`MM patient cells treated with lenalidomide or dexametha-
`sone as single agents enhanced Egr1, Egr2, and Egr3 ex-
`pression up to 1.5-fold compared with DMSO, whereas the
`combination synergistically enhanced Egr1, Egr2, and Egr3
`expression 2.5-, 2.7-, and 2-fold, respectively after 24 hour
`drug treatment (Fig. 2C). Similar results were seen in MM
`patient cells incubated for 6 hours (data not shown). Syner-
`gistic upregulation of Egr1 and p21 in LP-1 and JJN-3 cells,
`respectively (Fig. 3A), and p27 in NCI-H929 cells (Fig. 3B)
`was observed at the protein level.
`
`ALVOGEN, Exh. 1045, p. 0004
`
`

`

`Dexamethasone Synergizes with Lenalidomide
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`Current Cancer Drug Targets, 2010, Vol. 10, No. 2 159
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`Fig. (1). Combination of lenalidomide and dexamethasone causes synergistic inhibition of proliferation on multiple myeloma cell lines LP-1
`and OPM-2. (A) L P-1 cells were treated with 1.1 μM of lenalidomide alone, dexamethasone alone at nine concentrations with the high-
`est concentration 1 μM, and combination 1.1 μM of lenalidomide with dexamethasone at different concentrations for 96 hours. (B) LP-1
`cells were treated with 37 nM of dexamethasone alone, lenalidomide alone at nine concentrations with the highest concentration 100 μM, and
`combination 37 nM of dexamethasone with lenalidomide at different concentrations for 96 hours. (C) OPM-2 cells were treated with
`1.1 μM of lenalidomide alone, dexamethasone alone at nine concentrations with the highest concentration 1 μM, and combination 1.1
`μM of lenalidomide with dexamethasone at different concentratio ns for 96 hours. (D) OPM-2 cells were treated with 37 nM of dexametha-
`sone alone, lenalidomide alone at nine concentrations with the highest concentration 100 μM, and combination 37 nM of dexamethasone with
`lenalidomide at different concentrations for 96 hours. The y-axis is the fraction unaffected calculated as percentage of positive control. Lines
`shown are lenalidomide alone (black), dexamethasone alone (green), lenalidomide plus dexamethasone (blue), and the predicted additive
`effect (red).
`
`Table 3.
`
`
`Induction of Tumor Suppressor Genes in Myeloma Cell Lines
`
`Cell Line
`
`Time (hour)
`
`Lenalidomide
`
`Dexamethasone
`
`Combination*
`
`Karpas-620
`
`NCI-H929
`
`LP-1
`
`6
`
`24
`
`6
`
`24
`
`6
`
`24
`
`Egr3
`
`Egr1, p15, p21
`
`Egr2, Egr3
`
`-
`
`Egr2, Egr3
`
`p21
`
`-
`
`-
`
`Egr1, Egr2, p15
`
`-
`
`p21
`
`p21
`
`Egr1, p15
`
`Egr1, Egr3, p15, p21
`
`Egr1, p15, p21
`
`Egr3, p27
`
`p21
`
`p21
`
`ALVOGEN, Exh. 1045, p. 0005
`
`

`

`160 Current Cancer Drug Targets, 2010, Vol. 10, No. 2
`
`(Table 3). Contd.....
`
`Gandhi et al.
`
`Cell Line
`
`Time (hour)
`
`Lenalidomide
`
`Dexamethasone
`
`Combination*
`
`U266 B1
`
`JJN-3
`
`RPMI-8226
`
`Patient
`MM cells
`
`6
`
`24
`
`6
`
`24
`
`6
`
`24
`
`24
`
`-
`
`p21
`
`-
`
`Egr3, p15
`
`Egr3
`
`-
`
`-
`
`-
`
`Egr1
`
`Egr1
`
`p21
`
`-
`
`p21
`
`p21
`
`Egr1
`
`Egr1, Egr2, Egr3, p21
`
`Egr2, p21, p27
`
`-
`
`Egr1, Egr2, Egr3, p15, p16, p21, p27
`
`Egr1, Egr2, Egr3, p15, p16, p21
`
`Egr1, Egr2, Egr3, p21
`
`*Bold signifies synergistic effect, otherwise additive effect.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Fig. (2). Tumor suppressor gene induction at 24 hours in (A) LP-1, (B) JJN-3, and (C) multiple myeloma (MM) patient cells treated with
`lenalidomide (Len) and dexamethasone (Dex). *P < 0.05, **P < 0.01, ***P < 0.001 versus DMSO control determined by 2-way repeated
`measured analysis of variance followed by Bonferroni test. Data shown for LP-1 and JJN-3 cells are an average of 4 experiments, and pri-
`mary MM cell data is from 1 patient.
`
`ALVOGEN, Exh. 1045, p. 0006
`
`

`

`Dexamethasone Synergizes with Lenalidomide
`
`Current Cancer Drug Targets, 2010, Vol. 10, No. 2 161
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Fig. (3). Effect of lenalidomide, dexamethasone and combination of both drugs on the expression of cell cycle regulatory proteins. Cell
`lysates were prepared from (A) LP-1 and JJN-3 treated with DMSO or with different concentrations of lenalidomide or dexamethasone for 24
`hours. (B) NCI-H929 and LP-1 cells treated with DMSO or with different concentrations of lenalidomide or dexamethasone for 24 or 48
`hours followed by western blot analysis to detect the indicated proteins. Actin levels confirm similar protein loading.
`
`Lenalidomide and Dexamethasone Inhibit Retinoblastoma
`(Rb) Phosphorylation
`
`Upregulation of p21 by lenalidomide has been shown to
`inhibit CDK2, CDK4 and CDK6 kinase activity leading to
`the loss of Rb phosphorylation and the inability of MM cells
`to progress into S phase [2]. Dexamethasone has also been
`shown to inhibit Rb phosphorylation via p21 upregulation
`and CDK2 inactivation in rat hepatoma cells [12]. To exam-
`ine the effects of dexamethasone on lenalidomide-inhibited
`Rb phosphorylation, phospho-Rb protein analysis was done
`in NCI-H929 and LP-1 cells treated with lenalidomide alone,
`dexamethasone alone, or dexamethasone plus lenalidomide
`for 24 and 48 hours. After 48 hours, lenalidomide (1 (cid:1)M and
`10 (cid:1)M) and dexamethasone (1 (cid:1)M) as single agents inhib-
`ited Rb phosphorylation at S807/811 and S608, while the
`combination had an even greater inhibitory effect (Fig. 3B).
`These data suggest that the synergistic effects of lenalido-
`mide and dexamethasone on p21 upregulation are associated
`with greater inhibition of Rb phosphorylation in the presence
`of both drugs.
`
`Lenalidomide and Dexamethasone Enhance Caspase Acti-
`vation
`
`In order to understand the signaling pathways of le-
`nalidomide and dexamethasone-induced apoptosis in MM
`cells, the activation of caspase pathways in 7 MM cell lines
`
`treated with lenalidomide and dexamethasone alone, and in
`combination, was assessed by fluorescence-based caspase 3,
`8, and 9 activity assays. Lenalidomide alone activated
`caspase 3 in Karpas-620 and KMS-12-BM cells, caspase 8 in
`Karpas-620, KMS-12-BM, U266 B1 and RPMI-8226 cells,
`and caspase 9 in Karpas-620 and KMS-12-BM cells (see
`Table 4). Dexamethasone alone activated caspases 3 and 8 in
`Karpas-620, KMS-12-BM, NCI-H929, and JJN-3 cells, and
`caspase 9 in JJN-3 and RPMI-8226 cells. Lenalidomide plus
`dexamethasone synergistically induced caspase 3 activity in
`Karpas-620, NCI-H929, LP-1, U266 B1, and JJN-3 cells,
`caspase 8 activity in Karpas-620 and JJN-3 cells, and
`caspase 9 activity in Karpas-620, U266 B1, and JJN-3 cells.
`Each cell line had a distinct pattern of caspase activation
`with lenalidomide and dexamethasone. In Karpas-620 cells
`lenalidomide activated all three caspases up to 2-fold com-
`pared with DMSO, whereas dexamethasone had no effect
`(Fig. 4A). The drug combination synergistically activated
`caspases 3, 8, and 9 up to 2.5-fold compared with DMSO. In
`KMS-12-BM cells, lenalidomide alone induced caspases 3, 8
`and 9, and dexamethasone alone induced caspases 3 and 8
`(Fig. 4B). However, there was no synergistic activation of
`caspases in the KMS-12-BM cells. The protein kinase inhibi-
`tor staurosporine was included as a positive control.
`
`ALVOGEN, Exh. 1045, p. 0007
`
`

`

`162 Current Cancer Drug Targets, 2010, Vol. 10, No. 2
`
`Gandhi et al.
`
`Table 4.
`
`
`Induction of Caspase Activity in Myeloma Cell Lines
`
`Cell Line
`
`Time (hour)
`
`Lenalidomide
`
`Dexamethasone
`
`Combination*
`
`Karpas-620
`
`KMS-12-BM
`
`NCI-H929
`
`LP-1
`
`U266 B1
`
`JJN-3
`
`RPMI-8226
`
`24
`
`48
`
`24
`
`48
`
`24
`
`48
`
`24
`
`48
`
`24
`
`48
`
`24
`
`48
`
`24
`
`48
`
`*Bold signifies synergistic effect, otherwise additive effect.
`
`
`3, 8, 9
`
`3, 8, 9
`
`3, 8, 9
`
`3, 8
`
`-
`
`-
`
`-
`
`-
`
`8
`
`-
`
`-
`
`-
`
`8
`
`-
`
`-
`
`3, 8
`
`8
`
`3, 8
`
`-
`
`3, 8
`
`-
`
`-
`
`-
`
`-
`
`3, 8, 9
`
`3, 8, 9
`
`9
`
`-
`
`3, 8, 9
`
`3, 8, 9
`
`3
`
`3, 8
`
`-
`
`3, 8
`
`-
`
`3
`
`-
`
`3, 9
`
`3, 8, 9
`
`3, 8, 9
`
`8
`
`3
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Fig. (4). Caspase 3, 8, and 9 activation in (A) Karpas-620 at 24 hours and (B) KMS-12-BM cells treated with lenalidomide (Len) and dex-
`amethasone (Dex) at 48 hours. *P < 0.05, **P < 0.01, ***P < 0.001 versus DMSO control determined by 2-way repeated measured analysis
`of variance followed by Bonferroni test.
`
`Lenalidomide and Dexamethasone Induce G0/G1 Arrest in
`MM Cells
`
`To better understand the mechanism by which lenalido-
`mide and dexamethasone inhibit MM cell growth, cell cycle
`analysis was performed with each drug alone and in combi-
`nation (Fig. 5). G1 cell cycle arrest by lenalidomide in MM
`cells has been described previously [2]. Lenalidomide and
`dexamethasone alone induced 20–30% more G0/G1 cell cycle
`
`arrest than DMSO in the lenalidomide-sensitive LP-1 cells.
`The drug combination enhanced cell cycle arrest in a par-
`tially additive mechanism leading to G0/G1 cell cycle arrest
`in 90–95% of cells (45–50% more than DMSO). In le-
`nalidomide-resistant RPMI-8226 cells, dexamethasone in-
`creased G0/G1 cell cycle arrest by 15% compared with
`DMSO, but lenalidomide alone had no effect. Lenalidomide
`plus dexamethasone induced G0/G1 cell cycle arrest synergis-
`
`ALVOGEN, Exh. 1045, p. 0008
`
`

`

`Dexamethasone Synergizes with Lenalidomide
`
`Current Cancer Drug Targets, 2010, Vol. 10, No. 2 163
`
`tically in RPMI-8226 cells leading to cell cycle arrest in 80%
`of cells (20% more than DMSO).
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Fig. (5). Cell cycle arrest in LP-1 (lenalidomide-sensitive) and
`RPMI-8226 (lenalidomide-resistant) cells treated with lenalidomide
`(Len) and dexamethasone (Dex; dose 0.8–1.0 μM) for 72 hours.
`
`Lenalidomide and Dexamethasone Synergistically Induce
`Apoptosis of MM Cells
`
`The downstream effect of cell cycle arrest by lenalido-
`mide and dexamethasone was examined using lenalidomide
`and dexamethasone alone, and in combination, in MM cells
`using apoptosis assays. In lenalidomide-sensitive LP-1 cells,
`neither lenalidomide nor dexamethasone alone induced
`apoptosis, but the combination significantly and synergisti-
`cally induced apoptosis compared to either lenalidomide or
`dexamethasone alone (Fig. 6). In lenalidomide-resistant
`RPMI-8226 cells, neither lenalidomide or dexamethasone
`alone nor the combination induced apoptosis.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Fig. (6). I