Supporting Information - PNAS Information Beltran et al. 10.1073/pnas.1018941108 ... performed with 1–16 Gy using the Gammacell 1000 Elite/3000 Elan machine (MDS Nordion)

Supporting InformationBeltran et al. 10.1073/pnas.1018941108SI Materials and MethodsHuman Cell Lines and Patient Samples. Ten human-derived MCLcell lines—GRANTA519 (G519), HBL2, Z138, REC1, JVM2,UPN1, JEKO1, L128, MINO, and IRM2—were cultured understandard conditions. Biopsy specimens from 183 patients di-agnosed with MCL obtained from three series were analyzed: (i)a group of 68 Spanish MCL patient samples previously charac-terized by a-CGH and FISH (1); (ii) a set of 59 biopsy specimensfrom German MCL patients from Kiel University; and (iii) 56biopsy specimens from Spanish MCL patients collected at CNIO(Madrid, Spain) and deposited in a tissue microarray (TMA). Allcases presented with t(11;14)(q13;q32) translocation, CCND1-IGH gene fusion, and/or cyclin-D1 overexpression. For in vitrotherapeutic experiments using primary MCL samples, mono-nuclear cells were isolated from fresh peripheral blood of fourunselected patients with leukemic MCL. All clinical sampleswere obtained before initiation of therapy. The human inves-tigations were approved by an Institutional Review Board onScientific and Ethical Affairs.

Drug Screening.A cellular-genomic-proteomic approach was takento identify molecular predictors of therapeutic response to a panelof clinically relevant compounds targeting selected molecularpathways in human MCL-derived cell lines. Individual tumor cy-totoxicity profiles were correlated with genomic and gene ex-pression variations determined by a-CGH and gene expressionmicroarrays, respectively, and with proteomic analysis. The drugsused were ABT-737 and the negative control enantiomer com-pound, kindly provided by Dr. Saul Rosenberg (Abbott) (2);suberoylanilide hydroxamic acid (Vorinostat; Merck), trichostatinA (Sigma-Aldrich), roscovitine (A.G. Scientific), flavopiridol (sa-nofi-aventis), rapamycin (Wyeth), and bortezomib (MillenniumPharmaceuticals). In addition, individual mouse lymphomas weretreated with ABT-737, roscovitine, bortezomib, doxorubicin(Pfizer), and TW-37 (kindly provided by S. Wang and J. Chen,University of Michigan).

Quantitative Real-Time PCR. Total RNA was isolated from the celllines and patient samples by TRIzol extraction (Invitrogen).cDNA was generated with the M-MLV RT enzyme (Invitrogen).qRT-PCR was performed in triplicate with SYBR Green PCRMaster Mix (Applied Biosystems) and the ABI Prism 7500 system(Applied Biosystems). Results were normalized to GAPDH.CCND1-specific primers were forward, 5′-CAAACACGCGCA-GACCTTC-3′; reverse, 5′-CTGGAGAGGAAGCGTGTGAG-3′. Mouse GAPDH-specific primers were forward, 5′-ACTTTG-TCAAGCTCATTTCC-3′; reverse, 5′-TGCAGCGAACTTTA-TTGATG-3′. BCL2-specific primers were forward, 5′-GTCAA-CCGGGAGATGTCG-3′; reverse, 5′-GCATGCTGGGGCCG-TAC-3′. Murine mBcl2-specific primers were forward, 5′-GGA-TGACTGAGTACCTGAACC-3′; reverse, 5′-CAGCCAGGAG-AAATCAAACAG-3′.

Microarray Analysis of Human and Mouse Lymphomas. Genome-wideDNAcopynumber changesofhumanandmouse tumorswereanalyzed by a-CGH using the UCSF Human 2.0 and Mouse BACarrays, respectively, as described in detail previously (3). Geneexpression analysis of MCL cell lines was performed in duplicateusing the Affymetrix Gene Chip HG-U133 Plus 2.0 arrays on anin-house Affymetrix workstation, as reported previously (3). Todetermine the ABT-737 gene expression signature, those genesets with a statistically significant difference in expression (B > 0)

between the sensitive and resistant MCL cell lines were selectedand mapped to the genome using the Ensemble database (4). Astandard hypergeometric test was used for the enrichment anal-ysis, in which the genes represented in the microarray were takenas the reference set. Gene expression microarray data were sub-mitted to GEO (accession no. GSE25613).

siRNA Knockdown of CCND1 and Bax Genes. Human CCND1 andmurine Bax gene silencing was performed in human MCL celllines and mouse lymphomas, respectively, by transfecting 2 μMof two different siRNAs against CCND1 (Ambion) or BAX(Ambion), or using the Silencer Negative Control-1 siRNA(Ambion) with the Amaxa Nucleofector device, as describedpreviously (5). Cells were grown for 24 h before ABT-737treatment. Measurement of cyclin-D1 and Bax protein expres-sion was evaluated by Western blot analysis at 72 h.

Cell Viability, Apoptosis, and Cell Cycle Studies. Cell viability wasdetermined using the 3-(4,5-dimethyl-thiazol-2yl)-5-(3-carbox-ymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) re-duction assay (Promega).Apoptosiswas assessedusing theAnnexinV-FITC Apoptosis Detection Kit (BD Biosciences). Cell fluores-cence was detected on a FACSCalibur flow cytometer (BD Bio-sciences) and analyzed with CellQuest Pro software (BDBiosciences). In addition, activation of caspase 9 protein wasevaluated by Western blot analysis (Cell Signaling). For cell cycleevaluation, cells were collected by centrifugation, washed withPBS, fixed with 70% ethanol, incubated with 0.5 mg/mL RNase(Sigma-Aldrich) at 37 °C for 30 min, and stained with propidiumbromide. Cell fluorescence was evaluated by flow cytometry. Allexperiments were performed in triplicate after 48 h of incubationwith the corresponding drugs.

Western Blot and IHC Analyses.Westernblot studieswereperformedas described previously (6). Proteins were detected using the fol-lowing antibodies: BAX, caspase-9, P27Kip1, and P21Cip1 (CellSignaling); BID, BCL2 (human), BAD, BAK, BIM, and MCL1(human) (Stressgen); cyclin-D1, Bcl2 (mouse), MYC, RB, andP53 (Santa Cruz Biotechnology); actin (Calbiochem); P16INK4a

(Lab Vision); BCL-XL, Mcl1 (mouse), p19ARF (mouse), andCDK6 (Abcam); and CDK4 (BD Pharmingen). For the charac-terization of mouse lymphomas by IHC assays, the following an-tibodies were used: BCL2, cyclin-D1, P53, andKi67 (Dako). In thebiopsy specimens included in the TMA, IHC, and FISH analysesfor BCL2 expression and BCL2 gene copy number, respectively,were performed. The expression of BCL2 was measured by theARIOL semiautomated computerized training system, as reportedpreviously (7). The operation involves quantitative measurement(number of cells in a core of the patient’s sample) and qualitativeassessment (intensity of staining). Pearson’s correlation was ap-plied to BCL2 protein expression and BCL2 gene copy number.

Flow Cytometry Analysis of Murine Lymphomas. Cells from themurine IL-3–dependent BAF3 cell line, from clone CyD1-4, andfrom selected cyclin-D1–expressing lymphomas [CyD1-4–BCL2(tumor 268), CyD1-4–BCL2 (tumor 272), CyD1-4–1Gy (tumor15), and CyD1-4–1Gy (tumor 601)] were incubated with rat anti-mouse B220 APC (clone RA3-6B2), rat anti-mouse IgM FITC(clone R6-60.2), rat anti-mouse CD23 FITC (clone B3B4), andrat anti-mouse CD5 FITC (53-7.3) (BD Pharmingen; 1:100 di-lution). Cell fluorescence was detected using a FACSCalibur flowcytometer (BD Biosciences) and analyzed using FlowJo version7.6.1 software. All experiments were performed in duplicate.

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FISH Analysis. FISH was performed on fixed lymphoma cells fromcytogenetic analysis and on lymphoma biopsy specimens on theTMA as described previously (8, 9). A chromosome 10 centro-meric probe (Vysis CEP10 SpectrumOrange; Abbott Molecular)and a specific probe for BCL2 (Vysis LSI-BCL2 Dual-ColorBreak-Apart Probe; Abbott Molecular) were used. The BCL2copy number was calculated in relation to the tumor ploidymeasured by the number of CEP10 signals.

In Vivo Xenografted Mouse Models and Treatment with ABT-737. Allmouse experiments were performed in the Animal Core Facilitiesof the Center for Applied Medical Research (University ofNavarra) after being approved by the local Animal Ethics Com-mittee. Mouse xenografts were generated and monitored asreported previously (5). Groups of sixteen RAG2−/−γc−/− 6- to 8-wk-old mice (10) were inoculated i.v. via the tail vein with 2.5 ×106 cells from HBL2, G519, JEKO1, and L128 cell lines. For invivo therapy, mice received i.p. injections of ABT-737, which wasmixed with 30% propylene glycol, 5% Tween 80, and D5W (pH1.0), sonicated, and adjusted to pH 4–5. Four days after trans-plantation, eight mice from each group received ABT-737 (200μL i.p.) at 40 mg/kg/d for 14 consecutive days. The remainingeight mice received i.p. treatment with vehicle (control group).For survival analysis, time of death was considered to be thatoccurring either spontaneously or by elective killing because ofpain or suffering, in accordance with ethical criteria. OS was es-timated by the Kaplan–Meier method using GraphPad Prismversion 4.00. Tumor responses to the different therapies weremonitored in vivo by PET imaging (see below).

Generation of Cyclin-D1 Murine Models. A 1.1-kb fragment of hu-man CCND1 cDNA was amplified by PCR, digested withEcoRV, and ligated to the vector Combit-TA (kindly providedby Dr. I. Sanchez-Garcia) at the ScaI site (11). Combit-TA andCombit-TA-CCND1 plasmids were stably transfected into BaF3cells using the Amaxa Nucleofector device. Two cyclin-D1–ex-pressing clones were isolated by limiting dilution (termed CyD1-1 and CyD1-4), and 2.5 × 106 cells from each clone were in-oculated i.v. into RAG2−/−2γc−/− mice and monitored for tumordevelopment. To test whether irradiation can induce cell trans-formation, random irradiation of CyD1-1 and CyD1-4 cells wasperformed with 1–16 Gy using the Gammacell 1000 Elite/3000Elan machine (MDS Nordion). After irradiation, cells werecultured in media without IL-3. Several surviving clones wereidentified, expanded, and injected into immunocompromisedmice, which were followed up for tumor development. One ofthe clones that consistently generated tumors, the CyD1-4–1Gyclone, was selected for further experiments.To test whether coexpression of BCL2 could induce trans-

formation in cyclin-D1–expressing cells, the human BCL2 cDNAwas PCR-amplified from pCMV-Sport6 plasmid (RZPD;ImaGenes), digested with XhoI and EcoRI (New England Bi-olabs), and cloned into the pcDNA3.1 vector (Invitrogen). ThenBCL2 cDNA was subcloned into the pcDNA3.1/Hygro vector(Invitrogen) by ligating the NheI and HindIII (New EnglandBiolabs) fragment of pcDNA3.1-BCL2 into the pcDNA3.1/Hygro vector. Then pcDNA3.1/Hygro and pcDNA3.1/Hygro-BCL2 plasmids were stably transfected in CyD1-4 cells, andBCL2+ cells were selected with hygromycin. The CyD1-4 cellsexpressing BCL2 were injected i.v. (2.5 × 106) intoRAG2−/−γc−/− mice and monitored for tumor development (10).When signs of disease were observed in mice carrying eitherCyD1-4–1Gy or CyD1-4–BCL2 cells, spleens were harvested,and lymphoma cells were isolated and grown in culture mediumwithout IL-3. Independent CyD1-1–1Gy and CyD1-4–BCL2lymphoma cell lines were established and used for in vitro and invivo experiments. In vivo treatment of mouse lymphomas withDox and ABT-737 was performed after i.v. inoculation of 75 × 103

and 1 × 103 murine cells, respectively, into four groups of eightRAG2−/−γc−/− mice each. Four days after transplantation, eachgroup was treated with vehicle (i.p.), vehicle plus Dox (4 mg/mLin the drinking water), ABT-737 (200 μL i.p.) at 40 mg/kg/d for 14consecutive days, or ABT-737 plus Dox. For survival analysis, timeof death was considered to be that either occurring spontaneouslyor resulting from elective killing because of pain or suffering, inaccordance with ethical criteria. At the time of death, spleens andlivers were isolated and used for IHC and Western blot analyses.OS was estimated by the Kaplan–Meier method.

Generation and Bioluminescence Monitoring of the CyD1-4–mBcl2Mouse Model. Murine Bcl2 was cloned from murine pro-B-cellline BaF3 cDNA using the following primers: forward, 5′-CA-GATGAATTCCACCATGGCGCAAGCCGGGAG-3′; reverse,5′-ATCTGGTCGACTCACTTGTGGCCCAGGTATG-3′. A 0.7-kb EcoRI-SalI fragment was cloned in the pBABE-puro retro-viral vector (Addgene) digested with the same enzymes. Byretroviral infection, CyD1-4 cells were transduced with the SFG-nesTGL expression vector, which contains a fusion protein withGFP, luciferase, and HSV1-tk (12). To test the tumorigenecity ofCyD1-4 cell clones carrying the SFG-nesTGL vector, these wereinoculated in RAG2−/−γc−/−, which were followed up for morethan 6 mo without showing any signs of tumor development.Next, CyD1-4 cells carrying the SFG-nesTGL vector were ret-rovirally transduced with the pBABE-puro-mBcl2 vector. Allretroviral infections were performed in accordance with methodsreported previously (5). CyD1-4 cell with SFG-nesTGL andpBABE-puro-mBcl2 vectors were inoculated in RAG2−/−γc−/−mice, as described above. Lymphoma development was monitoredby bioluminescence imaging using the IVIS Imaging System(Xenogen). Toward this end, mice were anesthetized by i.p. in-jections of 40 μL of xilacin (Rompun 2%; Bayer) and ketamin(Imalgène 500; Merial) (1:9 dilution), and 150 mg/kg of Lucif-erin (firefly D-luciferin potassium salt; Xenogen) was injected i.p.Bioluminiscence images were obtained at 1 s and processed withLiving Image version 2.11 (Xenogen) and analyzed with IGOR(WaveMetrics). The color scale refers to 106 photons per secondper squared centimeter and squared radian.

PET Imaging of Tumors in Mice. To monitor tumor responses tochemotherapy in vivo, PET imaging was performed at certaintime points in a dedicated small animal Philips Mosaic PETsystem, with a 2-mm resolution, a 11.9-cm axial field of view, anda 12.8-cm transaxial field of view, as reported previously (5).

Determination of BAX Conformational Changes.Cells were fixed andpermeabilized using the Cytofix/Cytoperm Kit (BD Pharmigen)for 20 min at 4 °C. Cells were washed with Perm/Wash buffer(BD Pharmigen) and incubated with antibodies against BAX(YTH-6A7; Trevigen) or mouse-irrelevant IgG (M-5284; Sigma-Aldrich) for 20 min at room temperature. Samples were thenwashed, incubated with goat Alexa Fluor 488 antibody (In-vitrogen), and analyzed by flow cytometry on a FACSCaliburflow cytometer (BD Biosciences). All experiments were per-formed at 24 h in triplicate.

IF Studies.Cold methanol-fixed and permeabilized cells were usedfor IF studies. Cells were attached to polylisine-coated slides andincubated for 1 h in blocking solution and then overnight at 4 °Cwith the corresponding primary antibody, BAX (Cell Signaling),and cyclin-D1 (Santa Cruz Biotechnology). Goat secondary an-tibodies conjugated with red or green fluorophores (anti-rabbitAlexa Fluor 594 or anti-mouse Alexa Fluor 488) were usedat optimal dilutions for a 30-min incubation. Nuclei were con-trasted with DAPI (Abbott) and mounted with Vectashield(Vector Laboratories). IF images were captured using a Zeiss



Axio Imager 21 inverted microscope with epifluorescence, anddata were recorded digitally.

Subcellular Fractionation.Humanandmouse lymphomaswere lysedin an ice-cold solution containing 0.2% Nonidet P-40, 5 mM so-dium phosphate (pH 7.4), 50 mM NaCl, 150 mM sucrose, 5 mMKCl, 2mMDTT, 1mMMgCl2, 0.5mMCaCl2, and 0.1mMPMSF.The cytoplasmic fraction was collected after centrifugation oflysates at 1,000 × g for 10 min at 4 °C. The resulting pellet wasresuspended in 100 μL of the lysis solution without Nonidet P-40and loaded onto a cushion of a solution containing 30% (wt/vol)sucrose, 2.5 mM Tris-HCl (pH 7.4), and 10 mM NaCl. Aftercentrifugation at 1,000 × g for 10min at 4 °C, nuclei were collectedand extracted for 30 min at 4 °C with an ice-cold solution con-taining 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.4), and 300mM NaCl. After centrifugation of the extract at 10,000 × g for 10min at 4 °C, the supernatant was collected as the nuclear fraction.Western blot analyses for cyclin-D1, BAX, Lamin A/C (as a con-trol of nuclear fraction; Cell Signaling Technology), and β-tubulin(as a control of cytoplasmic fraction; Sigma-Aldrich) were per-formed in nuclear and cytoplasmic fractions.

Immunoprecipitation Studies. Cells were washed and lysed in lysisbuffer [150mMNaCl, 5mMEDTA, 0.5mMCaCl2, 0.2%NonidetP-40, 0.2% Tween-20, 10% glycerol, 25 mM Hepes (pH 7.4)]supplemented with complete protease inhibitor tablets (Roche)for 30 min at 4 °C. Immunoprecipitations were performed using

an anti–cyclin-D1 antibody (Santa Cruz Biotechnology) cross-linked to protein A agarose beads (Pierce) for 6 h at 4 °C. Todetect interaction with BAX, immunocomplexes were collectedby centrifugation, washed with lysis buffer, and subjected to 15%SDS/PAGE. Then separated proteins were transferred to PVDFmembranes (Millipore). Membranes were incubated with anti-BAX antibody (Cell Signaling) overnight at 4 °C. Immune com-plexes were visualized by enhanced chemiluminescence (Pierce)and exposed to X-ray film. Similar procedures were performed toconfirm the interaction of cyclin-D1 with CDK4 in human MCLcells and in murine lymphoma cells, using antibodies for humanCDK4 (BD Pharmingen) and murine Cdk4 (Upstate Bio-technology). To test the putative cyclin-D1 binding to apoptoticproteins in the murine lymphomas, the following antibodies wereused: Bak (Sigma-Aldrich), Noxa (Calbiochem), and Puma, Bim,and Bid (Cell Signaling).

Therapy of Primary MCL Samples. Mononuclear cells were isolatedfrom fresh peripheral blood cells obtained from fourMCLpatientswith leukemic disease by Ficoll–Paque gradient centrifugation.Cells (4 × 105) were then incubated in RPMI-1640 medium sup-plemented with 20% FBS and treated with 10 μM roscovitine and0–50 nMABT-737, singly or in combination, for 24 h. Cell viabilitywas quantified by the MTS assay. Expression of cyclin-D1 andBCL2 was measured by Western blot analysis in all cells beforeinitiation of therapy. All experiments were performed in triplicate.

1. Rubio-Moscardo F, et al. (2005) Mantle cell lymphoma genotypes identified with CGHto BAC microarrays define a leukemic subgroup of disease and predict patientoutcome. Blood 105:4445–4454.

2. Oltersdorf T, et al. (2005) An inhibitor of Bcl-2 family proteins induces regression ofsolid tumours. Nature 435:677–681.

3. Mestre-Escorihuela C, et al. (2007) Homozygous deletions localize novel tumorsuppressor genes in B-cell lymphomas. Blood 109:271–280.

4. Hubbard TJ, et al. (2009) Ensembl 2009. Nucleic Acids Res 37(Database issue):D690–D697.

5. Richter-Larrea JA, et al. (2010) Reversion of epigenetically mediated BIM silencingovercomes chemoresistance in Burkitt lymphoma. Blood 116:2531–2542.

6. Rubio-Moscardo F, et al. (2005) Characterization of 8p21.3 chromosomal deletions inB-cell lymphoma: TRAIL-R1 and TRAIL-R2 as candidate dosage-dependent tumorsuppressor genes. Blood 106:3214–3222.

7. Tracey L, et al. (2008) Somatic hypermutation signature in B-cell low-gradelymphomas. Haematologica 93:1186–1194.

8. Sanchez-Izquierdo D, et al. (2001) Detection of translocations affecting the BCL6 locusin B cell non-Hodgkin’s lymphoma by interphase fluorescence in situ hybridization.Leukemia 15:1475–1484.

9. Ventura RA, et al. (2006) FISH analysis for the detection of lymphoma-associatedchromosomal abnormalities in routine paraffin-embedded tissue. J Mol Diagn 8:141–151.

10. Traggiai E, et al. (2004) Development of a human adaptive immune system in cordblood cell–transplanted mice. Science 304:104–107.

11. Pérez-Mancera PA, et al. (2005) Cancer development induced by graded expression ofSnail in mice. Hum Mol Genet 14:3449–3461.

12. Ponomarev V, et al. (2004) A novel triple-modality reporter gene for whole-bodyfluorescent, bioluminescent, and nuclear noninvasive imaging. Eur J Nucl Med MolImaging 31:740–751.



10 HumanMCL cell lines

Drugs targeting essential regulatorypathways in cancer cells:

CDK-inhibitors:Flavopiridol, Roscovitine.

Histone deacetylase inhibitors:Trichostatin A, Vorinostat.

BH3-only mimetic: ABT-737.

Proteasome inhibitor:Bortezomib.

mTOR inhibitor:Rapamycin.

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Fig. S1. Cyclin-D1 inhibition enhances sensitivity to apoptosis in MCL cells. (A) Knockdown of cyclin-D1 by siRNA in the MCL cell lines JEKO1 and REC1 wasassociated with an accumulation of cells in the G1 phase of the cell cycle and moderate growth retardation. (B) A cellular-genomic-proteomics approach wastaken to identify molecular predictors of therapeutic response to a panel of clinically relevant compounds targeting selected molecular pathways in 10 humanMCL-derived cell lines. Individual tumor cytotoxicity profiles were correlated with genomic and gene expression variations determined by comparative ge-nomic hybridization to BAC microarrays (a-CGH), gene expression microarrays, and proteomic analysis. (C) Initial screens showed that the individual MCL celllines presented variable sensitivity to each of the drugs. (D) Evaluation of the therapeutic responses to roscovitine, vorinostat, bortezomib, flavopiridol, ra-pamycin, and trichostatin A in the ABT-737–sensitive versus ABT-737–resistant MCL subgroups demonstrated that none of these compounds had a similarcytotoxic profile as the BH3 mimetic. These data indicate that ABT-737 sensitivity was exclusive of a subgroup of MCL cell lines. (E) Treatment with ABT-737induced apoptosis in the sensitive cell lines (GRANTA519, MINO, HBL2, and Z138), as shown by annexin V staining, but not in the resistant tumors (JEKO1 and

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REC1). (F) ABT-737-treated mice transplanted with HBL2 cells experienced longer OS than mice treated with vehicle, accompanied by decreased lymphomaweights in spleen and liver. In contrast, mice carrying JEKO1 cells treated with ABT-737 or vehicle had a similar clinical outcome, and no statistically significantchanges were observed between spleen and liver weights. (G) Tumor responses were monitored by microPET. The maximum standardized uptake value(SUVmax) measured by microPET, corresponding to the glycolytic metabolism of tumor cells, was calculated for each tumor. Prolonged OS was accompaniedwith a decrease in the tumor glycolytic activity of HBL2-sensitive cells treated with ABT-737 compared with untreated cells (SUVmax, 2.64 vs. 0; P < 0.001; threemice per group). However, in the resistant JEKO1 cells, no significant changes in SUVmax were observed on ABT-737 exposure (1.96 vs. 2.55; P = 0.07; three miceper group). (H) The combination of cyclin-D1 silencing and ABT-737 (250 nM) exposure resulted in a statistically significant decrease in cell viability (Fig. 1D),increase in apoptosis, and accumulation of cells in the G1 phase in two ABT-737–resistant MCL cell lines.




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Fig. S2. Genomic amplification and overexpression of BCL2 conditions ABT-737 sensitivity. (A) ABT-737–sensitive and ABT-737–resistant tumors were dis-tinguished by a gene expression signature composed of 93 overexpressed genes, 13 of which (14%) were mapped to chromosome bands 18q21-q22 (hy-pergeometric test, P = 6.85 × 10−15), including the BCL2 gene. Those genes mapped to chromosome 18q21 are marked with an asterisk. (B) Representation ofthe whole-genomic profile determined by a-CGH analysis in the 10 MCL cell lines. Only the four ABT-sensitive MCL cell lines showed genomic amplification ofthe chromosome region 18q21, including the BCL2 gene loci. (C) JEKO1 cells were ectopically transfected with either BCL2 or with an empty vector, and thentreated with increasing doses of ABT-737. No changes in cell viability were observed after treatment. (D) Representation of the a-CGH studies of three MCLcases (P4, P5, and P7) showing high-level amplification of BCL2 gene (between four and six copies). (E) Quantification of BCL2 expression by IHC in the 56 MCLcases on the TMA showed that tumors with genomic gain/amplification at the BCL2 gene locus had a greater number of cells with BCL2 expression comparedwith nonamplified lymphomas.









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log 2

ratio

Chr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1617181920

Trisomy of 7p Gain of 3q21

Gain of 3q21Gain of 15q21-q25

del(10)(q21-q24) (PTEN)

CyD1-4 Cells

-2 -

1 0

1 2

log 2

ratio

HumanSyntenicregions


del(1)(p21-p22.3)

CyD1-4-1Gy (r601) Tumor-2

-1

0 1

2lo

g 2ra

tio


-2 -

1 0

1 2

log 2

ratio


-2 -

1 0

1 2

log 2

ratio


-2 -

1 0

1 2

log 2

ratio

-2 -

1 0

1 2

log 2

ratio


-2 -

1 0

1 2

log 2

ratio

Chr. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1617181920

Trisomy of 7p Gain of 3q21

Gain of 3q21Gain of 15q21-q25

del(10)(q21-q24) (PTEN)

CyD1-4 Cells

-2 -

1 0

1 2

log 2

ratio

HumanSyntenicregions


J

Fig. S3. Generation and characterization of cyclin-D1–expressing lymphomas in mice. (A) Representation of the Combit-TA vector, a tetracycline-regulatorysystem used for conditionally expressing CCND1/cyclin-D1. (B) Human CCND1 gene cloned into the Combit-TA vector was stably transfected into murine BaF3cells, and different single-cell clones isolated by limiting dilution were transplanted into RAG2−/−γc−/− mice. (C) In two of these cyclin-D1–expressing cell clones(CyD1-1 and CyD1-4), expression of cyclin-D1 could be turned off by adding Dox to the culture medium. Western blot analyses were performed at 48 and 72 h.(D) In vitro cyclin-D1 silencing of CyD1-4 cells was not associated with an independent growth of IL-3 and did not substantially modify cell cycle or apoptoticrates, but increased cell proliferation after 48 h. (E) Isolated single-cell clones at different concentrations (1–10 × 106 cells) were inoculated i.v. intoRAG2−/−γc−/− mice, but no tumor development was consistently observed after 270 d. (F) Isolated cell suspensions from CyD1-4–1Gy and CyD1-4–BCL2 lym-phomas were transplantable to secondary RAG2−/−γc−/− recipients, shortening the median latency period of tumor development with respect to parental cells(median OS, 17 ± 1 d and 19 ± 2 d, respectively). (G) Representative example of lymphomas developed in mice. (H) CCND1 expression was fourfold greater inCyD1-4–1Gy lymphomas compared with CyD1-4–BCL2 lymphomas. BCL2 (total) expression was twofold greater in CyD1-4–BCL2 lymphomas compared withCyD1-4–1Gy lymphomas. (I) Flow cytometry studies of the murine lymphomas revealed a CD19+B220+CD5−CD23−IgM− phenotype, corresponding to an im-mature B lymphocyte phenotype that resembled this of the originating BaF3 cells. (J) Whole-genome a-CGH analysis of mouse lymphomas identified genomicalterations common to human MCL, such as the gains of mouse chromosomes 6 and 9q (syntenic with gains of human chromosomes 3q21.1-q21.2, 7p11-p22.3,and 15q21-q25), and the deletions of 3q (syntenic with loss of 1p21-p22) and of chromosome 19 (including the loss of human chromosome 10q21-q24, whichharbors PTEN). Gains of 3q and 7p and loss of 1p21-p22 have been reported more frequently in blastoid/pleomorphic variants than in classic MCL. The UCSCGenome Bioinformatics Site (February 2009 version) was used (http://genome.ucsc.edu/cgi-bin/hgGateway?org=human).


http://genome.ucsc.edu/cgi-bin/hgGateway?org=human





Fig. S4. Cooperation of cyclin-D1 and BCL2 combined targeting to treat MCL. (A) Addition of Dox to the drinking water of RAG2−/−γc−/− control mice did notmodify cyclin-D1 expression in liver cells. Cells stained with the cyclin-D1 antibody correspond to normal mouse hepatocytes. Dox treatment did not change theexpression of cyclin-D1 in these cells, suggesting that it corresponds to the endogenous protein. (B) Changes in the cell cycle in mouse lymphomas treated withDox, ABT-737, or the combination treatment. (C) A detailed description of the differences in growth rate, cell cycle and apoptotic indices in mouse lymphomasafter Dox-induced cyclin-D1 silencing, ABT-737 treatment, and the combination of Dox and ABT-737, with the corresponding controls. Remarkably, simulta-neous cyclin-D1 silencing and ABT-737 exposure had a synergistic therapeutic effect that was more effective than the individual cyclin-D1 inhibition or ABT-737exposure alone, as demonstrated by the decreased cell viability and the increased apoptosis. (D) Incubation of the murine lymphomas with the BH3 mimeticTW-37, bortezomib, and doxorubicin after Dox-induced cyclin-D1 inhibition was not associated with the synergistic therapy observed between ABT-737 andcyclin-D1 silencing.



Fig. S5. Generation and characterization of the CyD1-4–mBcl2 mouse model. (A) Scheme showing the steps for the generation of the CyD1-4–mBcl2 mousemodel. By retroviral infection, CyD1-4 cells were transduced with SFG-nesTGL vector (triple modality reporter). To test the tumorigenecity of CyD1-4 cell clonescarrying the SFG-nesTGL vector, these were inoculated in RAG2−/−γc−/−, which were followed up for more than 6 mo with no signs of tumor development. Next,CyD1-4 cells with the SFG-nesTGL vector were transduced with the pBABE-puro-mBcl2 retroviral vector, and then inoculated and monitored for lymphomadevelopment by bioluminescence imaging using the IVIS Imaging System (Xenogen). (B and C) qRT-PCR analysis of human cyclin-D1 and murine Bcl2 expressionlevels in the murine lymphomas. (D) Western blot analysis of the murine lymphomas for proteins involved in MCL pathogenesis. (E–G) In vitro treatment ofCyD1-4–mBcl2 lymphomas. Tumor 4126 showed synergistic therapy on treatment with Dox and ABT-737, as demonstrated by a marked decrease in cell viability

Legend continued on following page



(E) and increase in apoptotic levels (F) without major changes in the cell cycle (G). Cyclin-D1 expression measured by Western blot analysis was silenced withDox in the lymphoma cells. (H) Flow cytometry analysis revealed an increase in the unbound BAX protein fraction after cyclin-D1 silencing in CyD1-4–mBcl2lymphomas. (I) Mice engrafted with CyD1-4–mBcl2 lymphomas received Dox, ABT-737, Dox plus ABT-737, or vehicle. Simultaneous therapeutic targeting ofcyclin-D1 and BCL2 was associated with a statistically significantly longer OS in engrafted mice in comparison with the group of mice treated with vehicle, Dox,or ABT-737 (P = 0.017). Fourteen mice were included in each group. (J) Lymphoma monitoring was performed by bioluminescence imaging at days +7 and +15after cell injection. Weaker signals were observed in the group treated with the combination therapy at day +15 compared with the other subgroups.

Fig. S6. Histopathological examination of CyD1-4–BCL2 lymphoma biopsy specimens after the different in vivo treatments. Changes in apoptosis and pro-liferation were detected in the lymphoma cells treated with the combination therapy, but not in the other subgroups. Similar results were observed in CyD1-4–1Gy lymphomas. HE, H&E staining.



BA

D

C

E

Dox+ABT-737

0

25

50

75

100

An

nex

inV

-Cel

ls(%

) p < 0.05CyD1-4-1Gy

Ctrol Dox ABT-737

Scramble siBAX

Dox+ABT-737

0

25

50

75

100

An

nex

inV

-Cel

ls(%

) p < 0.05CyD1-4-1Gy

Ctrol Dox ABT-737

Scramble siBAX

0

25

50

75

100

An

nex

inV

-Cel

ls(%

) p < 0.05CyD1-4-1Gy

Ctrol Dox ABT-737

ScrambleScramble siBAXsiBAX

0

25

50

75

100

p < 0.05

CyD1-4-BCL2

Ctrol Dox ABT-737 Dox+ABT-737

0

25

50

75

100

p < 0.05

CyD1-4-BCL2

Ctrol Dox ABT-737 Dox+ABT-737

Cyclin-D1

ACTIN

BAX

- - + + - - + + ABT-737

- + - + - + - + Dox

Scramble siBAX

Cyclin-D1

ACTIN

BAX

- - + + - - + + ABT-737

- + - + - + - + Dox

Scramble siBAX

Fig. S7. Cyclin-D1–BAX interaction in murine and human lymphomas. (A) Western blot analysis showed that cyclin-D1 silencing decreased phosphorylated RBprotein levels and increased P27kip expression in mouse lymphomas. (B) Silencing of cyclin-D1 in mouse lymphomas did not modify the expression of Bcl2, Mcl1,Bcl-xl, Bax, or Bak, as determined by Western blot analysis. (C) Cyclin-D1/Bax immune complexes did not include Bak, Noxa, Bim, Puma, and Bad in the murinelymphomas. (D) Immunoprecipitation of cyclin-D1 complexes revealed the presence of BAX in JEKO1 cells and in the murine lymphomas (Fig. 5D), as well asCDK4/Cdk4 protein, a well-known partner of cyclin-D1. (E) Silencing of Bax by siRNA in CyD1-4–BCL2 and CyD1-4–1Gy murine lymphomas was associated witha significant reduction of the therapeutic effect after Dox-induced cyclin-D1 inhibition and ABT-737 exposure. Silencing of cyclin-D1 and Bax was confirmed byWestern blot analysis. Experiments were performed in duplicate.



Table S1. Evaluation of BCL2 expression and BCL2 copy number status in a TMA including samples from patients with MCL, relatedto Fig. 3

Case no. TMA no. No. of objects in color 1 No. of objects in color 2 Median value Cytoplasmic score BCL2 genomic status

1 44 1,368 5,569 3,468.5 19.72 Gain/amplification1 104 17,258 17,218 17,238 50.06 Gain/amplification2 152 16,165 18,776 17,470.5 46.26 Gain/amplification3 4 14,147 15,313 14,730 48.02 Gain/amplification3 142 7,982 8,272 8,127 49.11 Gain/amplification4 58 15,333 17,710 16,521.5 46.40 Gain/amplification4 117 16,803 20,000 18,401.5 45.66 Gain/amplification5 71 13,355 14,797 14,076 47.44 Gain/amplification5 118 13,438 15,950 14,694 45.73 Gain/amplification6 8 12,872 13,779 13,325.5 48.30 Gain/amplification7 50 2,787 3,595 3,191 43.67 Gain/amplification7 100 11,976 12,922 12,449 48.10 Gain/amplification8 62 13,191 9,323 11,257 58.59 Gain/amplification8 111 15,275 11,978 13,626.5 56.05 Gain/amplification9 10 12,349 16,549 14,449 42.73 Gain/amplification9 137 13,491 15,255 14,373 46.93 Gain/amplification10 115 12,646 5,599 9,122.5 69.31 Normal11 66 666 2,461 1,563.5 21.30 Normal12 77 3,566 4,573 4,069.5 43.81 Normal12 131 18,535 17,458 17,996.5 51.50 Normal13 3 10,599 11,855 11,227 47.20 Normal13 141 17,016 9,053 13,034.5 65.27 Normal14 24 11,952 12,486 12,219 48.91 Normal15 34 8,529 8,328 8,428.5 50.60 Normal15 95 18,300 13,254 15,777 58.00 Normal16 45 6,113 6,272 6,192.5 49.36 Normal16 105 4,331 4,222 4,276.5 50.64 Normal17 56 9,202 6,296 7,749 59.38 Normal17 116 11,213 6,824 9,018.5 62.17 Normal18 25 6,359 7,050 6,704.5 47.42 Normal18 163 13,835 14,796 14,315.5 48.32 Normal19 35 16,497 15,872 16,184.5 50.97 Normal20 96 14,213 14,077 14,145 50.24 Normal21 46 10,495 7,413 8,954 58.61 Normal21 106 9,183 12,676 10,929.5 42.01 Normal22 124 8,512 9,510 9,011 47.23 Normal23 5 8,324 7,132 7,728 53.86 Normal23 143 13,468 13,139 13,303.5 50.62 Normal24 16 16,449 14,029 15,239 53.97 Normal24 154 12,994 13,520 13,257 49.01 Normal25 36 7,180 8,160 7,670 46.81 Normal25 97 6,450 7,671 7,060.5 45.68 Normal26 47 11,325 13,509 12,417 45.60 Normal26 107 5,954 14,986 10,470 28.43 Normal27 80 1,350 2,737 2,043.5 33.03 Normal27 134 16,249 19,149 17,699 45.90 Normal28 27 13,577 7,635 10,606 64.01 Normal28 165 12,946 14,069 13,507.5 47.92 Normal29 37 11,955 12,895 12,425 48.11 Normal29 87 12,852 15,686 14,269 45.03 Normal30 59 20,000 20,000 20,000 50.00 Normal30 108 17,062 17,499 17,280.5 49.37 Normal31 70 1,955 2,033 1,994 49.02 Normal32 81 16,189 17,136 16,662.5 48.58 Normal32 126 5,426 10,543 7,984.5 33.98 Normal33 7 265 1,497 881 15.04 Normal34 156 10,384 4,451 7,417.5 70.00 Normal35 82 5,582 9,384 7,483 37.30 Normal35 127 2,150 8,878 5,514 19.50 Normal36 18 17,207 9,802 13,504.5 63.71 Normal36 146 662 2,098 1,380 23.99 Normal37 28 14,472 10,792 12,632 57.28 Normal



Table S1. Cont.

Case no. TMA no. No. of objects in color 1 No. of objects in color 2 Median value Cytoplasmic score BCL2 genomic status

37 157 8,342 8,177 8,259.5 50.50 Normal38 39 6,209 4,868 5,538.5 56.05 Normal38 89 15,034 15,490 15,262 49.25 Normal39 61 5,815 6,784 6,299.5 46.15 Normal39 110 14,470 9,166 11,818 61.22 Normal40 9 13,210 16,424 14,817 44.58 Normal40 136 18,387 20,000 19,193.5 47.90 Normal41 19 3,211 3,808 3,509.5 45.75 Normal41 147 16,756 13,971 15,363.5 54.53 Normal42 29 12,842 12,394 12,618 50.89 Normal42 158 16,798 17,807 17,302.5 48.54 Normal43 40 11 11 11 50.00 Normal43 90 12,697 13,072 12,884.5 49.27 Normal44 73 15,447 12,904 14,175.5 54.48 Normal44 120 17,997 20,000 18,998.5 47.36 Normal45 84 14,735 13,328 14,031.5 52.51 Normal45 128 6,526 3,620 5,073 64.32 Normal46 20 15,866 17,003 16,434.5 48.27 Normal46 148 1,332 1,635 1,483.5 44.89 Normal47 30 15,786 12,181 13,983.5 56.45 Normal47 159 11,740 14,084 12,912 45.46 Normal48 41 9,539 10,880 10,209.5 46.72 Normal48 91 5,237 12,292 8,764.5 29.88 Normal49 63 14,066 15,760 1,4913 47.16 Normal49 112 7,929 9,594 8,761.5 45.25 Normal50 74 18,833 20,000 19,416.5 48.50 Normal50 121 13,034 15,142 14,088 46.26 Normal51 85 966 966 966 50.00 Normal51 129 13,182 14,288 13,735 47.99 Normal52 11 7,021 8,374 7,697.5 45.61 Normal52 138 12,770 16,979 14,874.5 42.93 Normal53 21 5,872 9,827 7,849.5 37.40 Normal53 149 14,214 16,959 15,586.5 45.60 Normal54 31 12,369 10,105 11,237 55.04 Normal54 160 16,005 20,000 18,002.5 44.45 Normal55 42 11,601 14,517 13,059 44.42 Normal55 92 13,654 16,246 14,950 45.67 Normal56 53 11,660 10,744 11,202 52.04 Normal56 102 10,184 13,332 11,758 43.31 NormalLymph node 64 3,872 5,358 4,615 41.95 Normal

Biopsy specimens from Spanish MCL patients were collected at CNIO (Madrid, Spain) and deposited in a TMA, constructed using a Tissue Arrayer (BeecherInstruments). These tumors were analyzed by IHC and FISH for BCL2 expression and for BCL2 gene copy number, respectively. The expression of BCL2 wasmeasured with the ARIOL semiautomated computerized training system. This procedure involves quantification (number of cells in a core of a patient’s sample)and qualitative (intensity of staining) assessment. In addition, BCL2 gene copy number was measured as described in Materials and Methods. BCL2 expressiondata and BCL2 gene copy number results were obtained for 56 cases. Most cases were studied in duplicate.



Table

S2.

Histopathological

andim

munophen

otypic

characteriza

tionofthecyclin-D

1–drive

nlymphomas

inmice,

relatedto

Fig.4

Sample

no.

Tumortype

Celltype

Lymphnodemorphology

Lymphnodenecrosis

Live

rmorphology

Live

rnecrosis

Mitosisper

high-power

objectivefield

Cy-D1

P27k

ipBCL2

Ki67

P53

r206

CyD

1-4–

1Gy

Largecell

Polymorphic

Absent

Perisinusoidal

Absent

182

11

100

5r212

CyD

1-4–

1Gy

trea

tedwithDox

Largecell

Polymorphic

Absent

Perisinusoidal

Absent

210

11

100

0

r211

7CyD

1-4–

Bcl2

Largecell

Polymorphic

Absent

Diffuse

Absent

81

22

100

30r211

4CyD

1-4–

Bcl2

trea

tedwithDox

Largecell

Polymorphic

Absent

Perisinusoidal

anddiffuse

Absent

210

12

100

20

r14

CyD

1-4–

1Gy

Largean

dinterm

ediate

cells

Polymorphic

Absent

Perisinusoidal

Absent

132

11

100

0

r15

CyD

1-4–

1Gy

Largean

dinterm

ediate

cells

Polymorphic

Absent

Perisinusoidal

Absent

132

11

100

10

r601

CyD

1-4–

1Gy

Largean

dinterm

ediate

cells

Polymorphic

Absent

Perisinusoidal

Absent

162

11

100

0

r272

CyD

1-4–

Bcl2

Largecell

Polymorphic

Absent

Perisinusoidal

anddiffuse

Absent

141

12

100

0

r268

CyD

1-4–

Bcl2

Largecell

Polymorphic

Absent

Perisinusoidal

anddiffuse

Absent

121

12

100

20

r269

CyD

1-4–

Bcl2

Largean

dinterm

ediate

cells

Polymorphic

Absent

Perisinusoidal

anddiffuse

Absent

191

22

100

5

IHCstudiesforcyclin-D

1,P2

7kip,an

dBCL2

aregraded

from

2(highly

intense)to

0(noex

pression);IHCan

alyses

forP5

3an

dKI67indicatethepercentageofpositive

lystained

cells.



Table

S3.

Pharmacologic

inhibitionofcyclin-D

1an

dBCL2

inhuman

MCLcelllin

esan

dprimarybiopsy

specim

ens,

relatedto

Fig.7

Trea

tmen

tJEKO1(R)(P

=0.02

)REC

1(R)(P

=0.01

)L1

28(R)(P

=0.01

)IRM2(R)(P

=0.01

)G51

9(S)(P

=0.01

)HBL2

(S)(P

=0.01

)CyD

1-4–

1Gytumor

(P<

0.00

1)CyD

1-4–

BCL2

tumor

(P=0.00

2)

Cellviab

ility,%

Control

100(±

0.6)

100(±

1.2)

100(±

0.6)

100(±

1)10

0(±

1)10

0(±

0.7)

100(±

0.1)

100(±

0.1)

Roscovitine

81(±

2.2)

71(±

6.1)

80(±

5.8)

43(±

1)57

(±3.7)

67(±

4.4)

55(±

5.7)

59(±

0.5)

ABT-73

782

(±2.9)

84(±

4.5)

89(±

6.7)

60(±

1.2)

75(±

2.5)

82(±

10.4)

81(±

5.4)

82(±

9.5)

Roscovitine+

ABT-73

723

(±1.1)

(P<

0.05

)25

(±2)

(P<

0.05

)59

(±6.5)

(P<

0.05

)34

(±1.3)

(P<

0.05

)42

( ±1.3)

(P<

0.05

)48

(±4.7)

(P<

0.05

)14

(±0.2)

(P=0.00

1)16

(±3)

(P=0.01

)

Trea

tmen

tJEKO1(R)(P

<0.05

)REC

1(R)(P

<0.05

)L1

28(R)(P

<0.05

)IRM2(R)(P

<0.05

)G51

9(S)(P

<0.05

)HBL2

(S)(P

<0.05

)CyD

1-4–

1GyTu

mor

(P=0.02

)CyD

1-4–

BCL2

Tumor

(P=0.01

)

Annex

inV

cells,%

Control

0(±

0.1)

0(±

11.5)

0(±

2)0(±

1.1)

0(±

±1.9)

0(±

2.2)

0(±

0.1)

0(±

0.1)

Roscovitine

14(±

1.7)

13(±

1.2)

7(±

1.9)

20(±

4.8)

18(±±

5)31

(±6)

20(±

4)15

(±0.2)

ABT-73

713

(±1.8)

10(±

1.6)

14(±

5)21

(±4)

23(±±

6)14

(±1)

25(±

0.8)

10(±

1.6)

Roscovitine+

ABT-73

771

(±0.6)

(P<

0.01

)52

(±5.8)

(P<

0.01

)47

(±4.8)

(P<

0.01

)60

(±6.4)

(P<

0.01

)53

(±±

7.2)

(P<

0.01

)52

(±10

)(P

<0.01

)68

(±1.6)

(P<

0.05

)62

(±0.1)

(P<

0.05

)

Rep

resentationofthech

anges

incellproliferationan

dap

optosisobtained

withroscovitine,

aloneorin

combinationwithABT-73

7,in

human

MCLcelllin

esan

din

mouse

lymphomas.N

otably,theco

mbinationof

roscovitinean

dABT-73

7dem

onstratedmarke

dtherap

euticactivity,decreasingcellsurvival

andinducingmassive

apoptosisin

alllymphomas

tested

.



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Supporting Information - PNAS Information Beltran et al. 10.1073/pnas.1018941108 ... performed with 1–16 Gy using the Gammacell 1000 Elite/3000 Elan machine (MDS Nordion)