Antimyeloma activity of heat shock protein-90 inhibition
Blood Mitsiades et al. 107: 1092

Supplemental materials for: Mitsiades et al, Vol 107, Issue 3, 1092-1100

SUPPLEMENTAL INFORMATION

Cell lines and primary tumor specimens. We studied a panel of previously established human MM cell lines and sublines (MM-1S, MM-1R, MM-1S-Bcl-2, MM-1S-Akt, MM-1S-TR13, RPMI-8226/S, RPMI-8226/Dox40, RPMI-8226/LR5, RPMI-8226/MR20, INA-6, S6B45, NCI-H929, OCI-My5, OPM-1, OPM-2, LP-1, ARD, ARK, ARP-1, EJM, K620, KMM1, U266, XG1, MM-AS, MM-SV) as well as primary MM tumor cells. The dexamethasone (Dex)-sensitive parental line MM-1S and its Dex-resistant subline MM-1R cells were kindly provided by Dr Steven Rosen (Northwestern University, Chicago, IL); the chemo-sensitive parental MM cell line RPMI-8226/S, and its chemo-resistant sublines RPMI-8226/Dox40 (doxorubicin-resistant), RPMI-8226/MR20 (mitoxantrone-resistant), and RPMI-8226/LR5 (melphalan-resistant) cells were kindly provided by Dr William Dalton (Lee Moffitt Cancer Center, Tampa, FL); the OCI-My5 cells were provided by Dr Meissner (University of Ontario, Toronto, Canada); the EJM, LP-1, KMM1, K620, OPM-1, and OPM-2 cells were provided by Dr Leif Bergsagel; INA-6 cells were provided by Renate Burger (University of Erlangen-Nuernberg, Germany); NCI-H929 and U266 cells were purchased from American Type Cell Culture (ATCC); MM-1S-myrAkt and MM-1S-Bcl-2 cells were established by stable transfection of MM-1S cells with constructs encoding for constitutively active myristoylated form of Akt and for Bcl-2, respectively. MM-1S-TR15 is a TRAIL/Apo2L-resistant subline established after 15 successive cycles of treatment of TRAIL/Apo2L-sensitive MM-1S parental cells with human recombinant form of TRAIL/Apo2L (N. Mitsiades, unpublished observation). MM-SAR-1 (also referred to as MM-SA-1) cells were primary MM tumor cells from a patient resistant to the proteasome inhibitor bortezomib (PS-341) (cells maintained in vitro resistance to PS-341). We also studied the B-lymphoblastoid cell lines ARH-77 and IM-9, the leukemic cell line NALM-6 (purchased from ATCC), as well as a panel of human lymphoma cell lines, including the diffuse large B-cell lymphoma cell lines DHL-4, DHL-6, DHL-7, DHL-8 and DHL-10 (kindly provided by Dr Margaret Shipp, Dana-Farber Cancer Institute Boston, MA). Human microvascular endothelial cells (HMVECs) were kindly provided by Dr Judah Folkman (Children’s Hospital, Boston, MA). All cells were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum, L-glutamine, penicillin, and streptomycin (Life Technologies), except for INA-6 and XG-1 cells, which were cultured in media supplemented with 20% FBS and 2.5 ng/mL of human recombinant IL-6; and HMVECs which were cultured in MVGS medium (Clonetics) supplemented with bFGF and VEGF.

Primary tumor cells from 24 newly diagnosed MM patients, as well as plasma cells from 5 individuals with MGUS and 5 healthy donors were obtained, following informed consent, from bone marrow (BM) aspirate samples, processed for red blood cell lysis with 0.86% ammonium chloride, and immunomagnetic separation-based positive selection of plasma cells with microbead-conjugated human anti-CD138 mAbs (MACS, Miltenyi Biotech) as in prior studies1. Purity (>95%) was assessed by both morphology and flow cytometry (Becton-Dickinson FACSort, Franklin Lakes, NJ). Plasma cells were identified by CD38 and CD45 expression, and forward and side scatter characteristics. Baseline prognostic features (including paraprotein type and level, 2m, hemoglobin, creatinine, and calcium levels, presence of bone disease, and percentage plasma cell infiltrate) and prospective clinical follow-up data (including treatment received, as well as progression free and overall survival) collected from MGUS and MM cases of this cohort have been previously reported1.

Antibodies and reagents. Geldanamycin (NSC 122750), the prototypic hsp90 inhibitor of the ansamycin family, the active geldanamycin analogs 17-allylamino-17-demethoxy-geldanamcyin (17-AAG, NSC 330507), NSC 255109 (17-Demethoxy-17-amino-GA) and NSC 683664, and the inactive geldanamycin analogs NSC 682300, NSC 683666 and NSC 210753 were obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, National Cancer Institute (Bethesda, MD); PS-341 (bortezomib) from Millennium Pharmaceuticals (Cambridge, MA) and IMIDs from Celgene Corp. (Warren, NJ). Other reagents were obtained as follows: the mouse anti-human IGF-1R neutralizing monoclonal antibody aIR3 and Apo2L/TRAIL from Calbiochem; polyclonal or mouse monoclonal antibodies for Bcl-2, Bcl-XL, A1/Bfl-1, Bax, hsp90, Raf, src, phosphorylated and total MEK1/2, IKK-a, PAK3 and GAPDH from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal antibody against RANKL from Chemicon; polyclonal or monoclonal antibodies against hsp90, Akt, p-Akt, IKK-, FKHRL-1 and its phosphorylated forms, Raf-1, and p70S6K from Cell Signaling Technologies (Beverly, MA); polycloncal antibody against Bmx and monoconcal APC-conjugated antibody against CD45RA from BD Pharmingen (San Diego, CA); human recombinant IGF-1, IL-6, VEGF and bFGF, polyclonal antisera against cIAP-1, cIAP-2, and XIAP, as well as PE-conjugated monoclonal antibodies against IGF-1R, IL-6R, CD40, CD138, and FITC-conjugated antibody against CD38 from R&D Systems, Inc. (Minneapolis, MN); MTT, dexamethasone and doxorubicin from Sigma Chemical Co. (St Louis, MO); and the Enhanced Chemiluminescence (ECL) kit, which includes the peroxidase-labeled anti-mouse and anti-rabbit secondary antibodies, from Amersham (Arlington Heights, IL).

Transfections and retroviral transductions. Stable transfections of RPMI-8226/S cells with plasmid vector for Green Fluorescent Protein, GFP (Clontech) and of MM-1S cells with vectors encoding myristoylated (constitutively active) Akt or Bcl-2 (Upstate Biotechnologies, Lake Placid, NY) or with empty (neo) vectors, were performed, as previously described2,3. Retroviral transduction of MM-1S and MM-1R cells with a pGC-gfp/luc vector (kind gift of C.G. Fathman, Stanford University) utilizing Retronectin (Takara) (or 4 µg/mL polybrene (Sigma)) was performed as previously described4-6.

Ex vivo drug sensitivity assays. Cell survival of 17-AAG-treated cells was examined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) colorimetric assay, as previously described7.

In vivo anti-tumor activity of hsp90 inhibition. The in vivo anti-MM activity of 17-AAG was evaluated in a previously established model of diffuse GFP+ MM lesions in SCID/NOD mice by serially monitoring with whole-body fluorescence imaging8. Briefly, 40 male (6 to 8-week old) SCID/NOD mice were obtained from Jackson Laboratories (Bar Harbor, ME); housed and monitored in the Animal Research Facility of the Dana-Farber Cancer Institute; gamma-irradiated (300 rads) using 137Cs -irradiator source; and received (24 hours post-irradiation) tail i.v. injections of 5 × 106 RPMI-8226/S-GFP+ cells suspended in total volume of 100 µL of phosphate-buffered saline (PBS) per mouse. Mice were monitored daily for changes in body weight, signs of infection or paralysis, and with thrice weekly fluorescence imaging. In accordance with institutional guidelines, mice were sacrificed by CO2 inhalation in the event of paralysis or major compromise in their quality of life. All experimental procedures and protocols had been approved by the Animal Care and Use Committee of the Dana-Farber Cancer Institute.

Whole-body real-time fluorescence imaging. The development of fluorescent MM lesions in the skeleton and extra-skeletal sites was serially monitored by whole-body fluorescence imaging using the LT-9500 fluorescent light box (Lightools Research, Encinitas, CA), as previously described8. Briefly, fluorescence excitation of GFP tumors was produced through a 440±20 nm interference filter (excitation filter) using slit fiber optics for animal illumination. Fluorescence was observed through a 520-nm long pass filter (viewing filter). Fluorescence imaging results were digitally captured on a FujiFilm FinePix 6800Z digital camera (Fuji, Japan) and analyzed with Adobe Photoshop 7.0.

Fluorescence imaging-guided necropsy. During necropsy of sacrificed mice, fluorescence imaging-guided macroscopic inspection of internals organs was performed after generation of skin flaps and preparation of the spine, lung, liver, and spleen, and prior to further processing for histopathologic analysis. Samples from spine, skull, pelvis, extremities, thoracic cage, subcutaneous tissue or any other organs harboring fluorescing lesions (as well as control samples obtained from sites adjacent to fluorescent lesions or from unaffected contra-lateral sites) were processed for conventional histopathological examination.

Histopathologic analysis. Immediately after fluorescence imaging-guided necropsy, specimens from tissues with fluorescent lesions, and representative samples of non-fluorescent areas of several organs (including lungs, liver, spleen) were processed as previously described8-10.

In vivo molecular profile of hsp90 inhibition in MM cells. To confirm that molecular sequelae of hsp90 inhibition are consistent ex vivo and in vivo, we characterized the impact of 17-AG treatment of MM-bearing SCID/NOD mice on expression of hsp90 targets in MM cells. Sublethally irradiated SCID/NOD mice were injected intravenously with 5 × 106 MM-1S cells retrovirally transduced with construct for GFP/luciferase fusion gene (MM-1S-GFP+/luc+ cells). Similarly to the SCID/NOD model of GFP+ MM skeletal lesions established with RPMI-8226/S-GFP+ cells, MM-1S-GFP+/luc+ cells formed diffuse MM bone lesions primarily located in the axial skeleton (e.g. spine, skull, pelvis, etc.) with anatomic distribution and pathophysiologic sequelae (e.g. hind-limb paralysis due to vertebral lesions) consistent with clinical manifestations of the disease in MM patients8 and our in vivo studies evaluating the anti-tumor activity of 17-AAG against RPMI-8226/S cells. The spatio-temporal progression of MM lesions was serially monitored with whole-body fluorescence imaging. Three weeks after the injection of GFP+ tumor cells, a cohort of 7 SCID/NOD mice received single i.p. injections of either 17-AAG (50 mg/kg i.p.) or equal volumes of vehicle. Twenty-four hours after injections, mice were sacrificed by CO2 inhalation and necropsy was performed under the guidance of whole-body fluorescence imaging to identify the anatomic sites of GFP+ MM lesions. Skeletal areas encompassing such fluorescent MM lesions were excised; cellular infiltrates were flushed from marrow cavities with cold 1x PBS; and MM cells were purified by flow cytometric cell sorting (FACS) on the basis of their GFP+ status. The purified (>95% GFP+ CD138+) MM cell population which had homed to BM of SCID/NOD mice was then processed for molecular profiling studies, as described in separate sections of this supplement.

In vitro and in vivo gene expression profiling of hsp90 inhibition. The gene expression profiles of hsp90 inhibitor-treated MM cells were evaluated according to previously described protocols2. Briefly, total RNA was extracted and purified with the Qiagen RNeasy kit (Qiagen, San Diego, CA). Five micrograms of total RNA was used in the first-strand cDNA synthesis with T7-d(T)24 primer (GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24) and Superscript II (GIBCO-BRL, Rockville, MD). The second-strand cDNA synthesis was carried out at 16°C by adding Escherichia coli DNA ligase, E. coli DNA polymerase I, and RNase H to the reaction, followed by T4 DNA polymerase to blunt the ends of newly synthesized cDNA. The cDNA was purified through phenol/chloroform and ethanol precipitation. Using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY), the purified cDNA was incubated at 37°C for 5 h in an in vitro transcription reaction to produce cRNA labeled with biotin. cRNA (20 µg) was fragmented by incubating in a buffer containing 200 mM Tris-acetate (pH 8.1), 500 mM KOAc, and 150 mM MgOAc at 94°C for 35 min. The hybridization cocktail containing 15 µg adjusted fragmented cRNA mixed with Eukaryotic Hybridization controls (contains control cRNA and oligonucleotide B2) was hybridized with a pre-equilibrated human U133A Affymetrix chip at 45°C for 16 h. After the hybridization cocktails were removed, the chips were washed in a fluidic station with low-stringency buffer (6 X standard saline phosphate with EDTA, 0.01% Tween 20, and 0.005% antifoam) for 10 cycles (two mixes/cycle) and high stringency buffer (100 mM N-morpholino-ethanesulfonic acid (MES), 0.1 M NaCl, and 0.01% Tween 20) for four cycles (15 mixes/cycle) and stained with SAPE (streptavidin phycoerythrin). This process was followed by incubation with normal goat IgG and biotinylated mouse anti-streptavidin antibody and restaining with SAPE. The chips were scanned in an HP ChipScanner (Affymetrix Inc, Santa Clara, CA) to detect hybridization signals. Scanned image output files were visually examined for major chip defects and hybridization artifacts and then analyzed with Affymetrix GeneChip Microarray Analysis Suite 5.0 software (Affymetrix). The image from each GeneChip was scaled such that the average intensity value for all arrays was adjusted to a target intensity of 150. The expression analysis files created by GeneChip Microarray Analysis Suite 5.0 software were exported as flat text files to Microsoft Excel and used for further analysis. Data analysis was performed to identify signals that were at least two-fold different between 17-AAG treated samples and their respective controls. These results were screened for p-values less than 0.0025 in Student’s t test, to identify transcripts that were induced or repressed. For hierarchical clustering analysis, data were imported into the Gene Cluster and TreeView software (Stanford University, Stanford, CA). The separation ratio was set at 0.99. Additional softwares used for data mining include GeneSpring 4.2.1 (Silicon Genetics). Data were visualized using the Rainbow program (developed by Charles Bailey and Towia Libermann) that enables representation of data in color format according to their values on a logarithmic scale. Annotations and informations for all genes were retrieved using the NetAffx Web site (www.affymetrix.com/analysis/index.affx) and UnChip (unchip.org:8080/bio/unchip; Alberto Riva, Atul Butte, and Isaac Kohane; Childrens Hospital, Boston) and added to the data file. Annotated data were sorted according to functional relationships.

Gene expression profiling of primary specimens from MM, MGUS patients and healthy donors. Gene expression profiling of primary specimens from MM, MGUS patients and healthy donors was performed according to previously described protocols1. Briefly, RNA was extracted using commercially available kits (Qiagen and Stratagene), according to manufacturers’ instructions. Following DNAse treatment, an Abl PCR was performed to confirm the absence of DNA contamination. The amount of RNA isolated was estimated by a real-time quantitative PCR (RQ-PCR) assay using a Pre-Designed Assay Reagent (PDAR) (Applied Biosystems) and a standard curve. In order to have adequate material for hybridisation, the starting RNA was amplified using a modified SMART™ PCR protocol (BD Biosciences, CA), in which a 5´ T7 polymerase promoter site was incorporated to create amplified cDNA compatible with downstream processing for the Affymetrix GeneChip® system. Briefly 0.05-1µg total RNA was combined with an oligo dT-T7 RT primer and template switch oligonucleotide prior to first strand synthesis (20nmoles Dithiothreitol, 10nmoles dNTP with PowerScript reverse transcriptase, BD Bioscience) at 42°C for 1hr. The resulting first strand SMART cDNA was then combined in a reaction containing 0.4mM dNTPs, 1.5mM MgCl2, 1× PCR buffer, 0.1µM T7 PCR primer, and 0.1µM SMART PCR primer, according to manufacturers instructions. Following a hot start (TaKaRa LA Taq, 1 min 95°C), thermal cycling conditions for the appropriate number of cycles were: 95°C for 5 sec, 65°C for 5 sec, and 68°C for 6 min (MJ DNA Engine thermal cycler). As PCR inevitably favours short sequences over long ones, it was necessary to assess the optimum number of PCR cycles (15, 18, 21, 24 or 27) for each sample so that the reaction could be terminated prior to over-cycling of the shorter sequences. The optimum cycle number was determined to be one cycle less than when the products became visible on 1.2% agarose EtBr gel. Following thermal cycling, the PCR products were cleaned up using the QiaQuick PCR Purification Kit (Qiagen), per manufacturer’s instructions. A further Abl PCR was performed to re-confirm the absence of contaminating genomic DNA. Affymetrix Human Genome U95AV2 GeneChip® arrays (Santa Clara, CA) containing probes for 12,600 expressed sequences were used for mRNA expression profiling. Biotinylated RNA was synthesized using the BioArray RNA transcript labeling kit (Enzo, Farmingdale, NY) with biotinylated ribonucleotides for 5 h at 37°C. In-vitro transcription products were purified using RNeasy columns (Qiagen, Valencia, CA). Biotinylated RNA was fragmented for 35 min at 94°C in 40mM Tris acetate (pH 8.1), 100mM potassium acetate, and 30mM magnesium acetate. Arrays were hybridized with biotinylated in-vitro transcription products for 16h at 45°C according to manufacturer’s instructions. Fluidic station 400 (Affymetrix) was used for washing and a three-step staining protocol was used to enhance detection of the hybridized biotinylated RNA: incubation with streptavidin-phycoerythrin conjugate, labeling with an anti-streptavidin goat biotinylated antibody (Vector Laboratories, Burlingame, CA), followed by staining with streptavidin-phycoerythrin conjugate. Arrays were scanned by the Affymetrix fluorescence reader (Hewlett Packard). The excitation source was an argon ion laser, and emission was detected by a photomultiplier tube through a 570-nm long pass filter. The raw image DAT data files were initially processed using Affymetrix GeneChip® software (version 4) to create CEL files for data clean-up and higher level analysis using DNA-Chip Analyzer. Array normalization and expression value calculation was performed using DNA-Chip Analyzer (dChip)11, which is freely available to academic users at www.dchip.org. The Invariant Set Normalization method11 was used to normalize arrays at probe cell level to make them comparable, and the model-based method11 was used for probe-selection and computing expression values. The standard errors with attached expression levels were subsequently used to compute the 90% lower confidence bound of fold change as a conservative estimate of the real fold change11.

Flow cytometry, immunoblotting analyses, and functional assays for telomerase, proteasome and transcription factor activities. Previously published experimental protocols were used for: flow cytometric analyses for IGF-1R, IL-6R and CD40 using an EPICS-XL-MCL flow cytometer (Beckman Coulter)7,12,13; immunoblotting analyses7; telomerase activity assay14,15; and 20S proteasome chymotryptic activity assay16. The DNA binding activities of NF-B and HIF-1 in hsp90 inhibitor-treated MM-1S cells were quantified by enzyme linked immunosorbent assay (ELISA) using Trans-AM™ Transcription Factor Assay Kits (Active Motif, Encinitas, CA) for NF-B p65 and HIF-1 activity, respectively. The use of ELISA formats for quantification of DNA binding activity of NF-B and HIF-1 (on immobilized oligonucleotides containing consensus binding sites for the respective transcription factors) has been previously substantiated in both MM and other models2,12,17,18. Nuclear vs. cytoplasmic extracts were prepared as previously reported19.

Proteomic analyses of signaling state of MM cells. High-throughput global proteomic analysis of the signaling state of hsp90 inhibitor-treated MM cells was performed by multiplex-immunoblotting arrays, as previously described18,20,21.

Statistical analyses. Statistical significance for the in vitro assay results was examined by a 2-way analysis of variance, followed by Duncan’s post-hoc test. In all analyses, p<0.05 was considered statistically significant. For assessment of in vivo anti-tumor activity, Kaplan-Meier survival analysis was performed to compare the overall survival of in the 17-AAG vs. control cohort (Overall survival of mice was defined as the time between i.v. injection of tumor cells and sacrifice or death).

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18. Mitsiades N, Mitsiades CS, Richardson PG, et al. The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications. Blood. 2003;101:2377-2380.

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21. Mitsiades CS, Mitsiades NS, McMullan CJ, et al. Inhibition of the insulin-like growth factor receptor-1 tyrosine kinase activity as a therapeutic strategy for multiple myeloma, other hematologic malignancies, and solid tumors. Cancer Cell. 2004;5:221-230.

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