Bcl-2 Family Genetic Profiling Reveals Microenvironment ...the beads by using EDC in a pH 4.5 MES...

Therapeutics, Targets, and Chemical Biology

Bcl-2 Family Genetic Profiling Reveals Microenvironment-Specific Determinants of Chemotherapeutic Response

Justin R. Pritchard1, Luke A. Gilbert1, Corbin E. Meacham1, Jennifer L. Ricks1, Hai Jiang1,Douglas A. Lauffenburger1,2, and Michael T. Hemann1

AbstractThe Bcl-2 family encompasses a diverse set of apoptotic regulators that are dynamically activated in response

to various cell-intrinsic and -extrinsic stimuli. An extensive variety of cell culture experiments have identifiedeffects of growth factors, cytokines, and drugs on Bcl-2 family functions, but in vivo studies have tended to focuson the role of one or two particular members in development and organ homeostasis. Thus, the ability ofphysiologically relevant contexts to modulate canonical dependencies that are likely to be more complex has yetto be investigated systematically. In this study, we report findings derived from a pool-based shRNA assay thatsystematically and comprehensively interrogated the functional dependence of leukemia and lymphoma cellsupon various Bcl-2 family members across many diverse in vitro and in vivo settings. This approach permitted usto report the first in vivo loss of function screen for modifiers of the response to a front-line chemotherapeuticagent. Notably, our results reveal an unexpected role for the extrinsic death pathway as a tissue-specific modifierof therapeutic response. In particular, our findings show that particular tissue sites of tumor dissemination playcritical roles in demarcating the nature and extent of cancer cell vulnerabilities and mechanisms of chemore-sistance. Cancer Res; 71(17); 5850–8. �2011 AACR.

Introduction

Chemotherapy represents a major treatment modality forcancer, and numerous genetic screens have probed themechanisms underlying cell-intrinsic resistance or sensitivityto front-line chemotherapy (1–6). However, these studies,although informative, have not been adapted to relevanttumor microenvironments, which may contain diverse stro-mal and/or immune cell types, are subject to immune sur-veillance, and harbor physical barriers to drug delivery (7). Inaddition, the native tumor microenvironment comprises adiverse mixture of chemokines and cytokines that may impactresponses to genotoxic agents (8, 9). Thus, the central deter-minants of therapeutic outcome may be highly dependentupon paracrine survival or stress signals. Indeed, it is welldocumented that gene function and relevance can vary dra-matically when compared in vivo versus in vitro (8, 10).Consequently, studying the impact of defined genetic altera-

tions on therapeutic response in native tumor microenviron-ments is critical for effective drug development, personalizedcancer regimens, and the rational design of combinationtherapies.

Recent advances in the development of tractable mousemodels of cancer have, for the first time, enabled the exam-ination of complex sets of defined alterations in individualmice. For example, retroviral infection of murine hematopoie-tic stem cells or primary embryonic hepatocytes with smallpools of short hairpin RNAs (shRNA), followed by adoptivetransfer into lethally irradiated recipient mice, has been usedto screen for suppressors of B cell lymphomagenesis orhepatocellular carcinoma (11, 12). In addition, ex vivo manip-ulation of lymphoma cells followed by transfer into syngeneicrecipient mice has permitted the interrogation of thousands ofshRNAs for modulators of tumor growth and dissemination(13). These screens provide powerful proofs of principle thatdiverse alterations can be introduced in chimeric tumormodels in vivo and that these systems might permit thesimultaneous examination of the relevance of a whole setof genes to therapeutic response in relevant physiologiccontexts.

Front-line cancer therapies generally exert their effects bymodulating the proportion of pro- to antiapoptotic deathregulators, most notably members of the Bcl-2 family (14,15). Thus, we reasoned that interrogating Bcl-2 family func-tionality might provide a high-resolution focus on a crucialfacet of cytotoxic cellular responses to chemotherapy in avariety of distinct settings. Notably, previous studies usingrecombinant BH3 peptides in reconstituted mitochondrial

Authors' Affiliations: 1The Koch Institute for Integrative Cancer Research;and 2Department of Biological Engineering, Massachusetts Institute ofTechnology, Cambridge, Massachusetts

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

J.R. Pritchard and L.A. Gilbert contributed equally to this work.

Corresponding Author: Michael T. Hemann, The Koch Institute forIntegrative Cancer Research, MIT, 77 Massachusetts Ave., 76-361B,Cambridge, MA 02139. Phone: 617-324-1964; Fax: 617-252-1891; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-11-1014

�2011 American Association for Cancer Research.

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suspensions have systematically identified cellular states asso-ciated with the loss of function of one of the BH3-only Bcl-2family members, the loss of function of a multidomain proa-poptotic Bcl-2 family member, or the enhanced function of anantiapoptotic family member; these states characterize thepotential range of dysregulation that the Bcl-2 family canacquire during tumorigenesis and demarcate central cell fatedecisions that are susceptible to therapeutic intervention (16,17). However, this approach, although quite powerful, does notallow for the comprehensive examination of the role andrelevance of individual Bcl-2 family members to cell deathfollowing chemotherapy. Here, we describe a complementaryin vivo screening approach that provides a detailed assess-ment of the role of each Bcl-2 family member in the responseto chemotherapy in heterogeneous tumor environments.

Materials and Methods

shRNA generationshRNAs targeting the Bcl-2 family (18) were designed by

using Biopredsi from Novartis. shRNAs were cloned into theMLS (19) retroviral vector containing a Mir30 expressioncassette under the transcriptional control of the MSCV LTRand coexpressing green fluorescent protein (GFP). Plasmidswere verified for mRNA target knockdown by using standardquantitative reverse transcriptase PCR techniques. Westernblots were done to analyze total protein knock down for asubset of Bcl-2 family members. Bim, Bax, and Bak knockdownare shown in Supplementary Figures S1A and B. The shRNAlibrary was constructed by evenly pooling individual mini-preps of each individual shRNA. This mixture was cotrans-fected into Phoenix retroviral packaging cells and pooled viruswas collected.

Western blotsSDS-PAGE was done according to standard protocols, and

gels were transferred to polyvinylidene difluoride membranes.The antibodies used were as follows: Bid (polyclonal antiserafrom Honglin Li), BAX (Cell Signaling Technologies; #2772),BAK (Upstate; #06-536), BIM (Cell Signaling Technologies;#C34C5), caspase 8 (Cell Signaling Technologies; #D35G2),beta-actin (Cell Signaling Technologies; #4967L), and Tubulin(ECM Biosciences; #TM1541).

Luminex bead–based assayCarboxylated Luminex beads were purchased from Mirai

Biosystems. Probe oligonucleotides comprised of the shRNAantisense strand modified with C12-amine were conjugated tothe beads by using EDC in a pH 4.5 MES hydrate buffer.Coupling efficiency was validated with sense oligonucleotides.A total of 3,500 beads were added to a 50-mL reaction volumein a 3 mol/L tetra-methyl ammonium chloride (TMAC) bufferto reduce the differences in Tm that accompany differences inGC content. DNA loading concentrations are as indicated (2–200 ng per well were added). Blocking oligos that corre-sponded to the biotinylated PCR product but lacked the senseportion of the shRNA were added at 100-fold molar excess tocompete out the dsPCR product and reduce the preferential

rehybridization of the PCR product. Samples were denaturedfor 3 minutes at 95�C and hybridized for 30 minutes at 52�C.Streptavidin-PE (Invitrogen)/TMAC was then added to thewells and incubated for 5 minutes at 52�C. All samples wereincubated at 52�C to ensure they stayed at equilibrium, andthe PMT setting on the Luminex machine was approximately520 volts (low calibration).

PCRThree individual 25 mL PCR reactions were done by using a

50 primer targeting the constant hairpin loop region and a 30

primer targeting the vector backbone. The PCR buffer was 2�Failsafe Buffer B (Epicentre Biotechnologies), and we ran 35cycles with an extension time of 1 minute at 72�C and ahybridization temperature of 52�C for 35 seconds. The 30

primer was biotinylated. Pooled PCR product was columnpurified and resuspended in 36 mL water prior to serialdilution and subsequent measurement.

Cell cultureAll lymphoma and leukemia cells were isolated directly

from tumor-bearing animals and cultured at 5% CO2 at 37�C.Em-Myc p19ARF�/� cells were isolated and maintained asdescribed (5). B-ALL cells were maintained as described(20). All in vitro viral transduction was done by coinfecting1 million cells per 10-cm plate. Infection efficiency wasquantified by flow cytometry for GFP-positive cell popula-tions. Infection efficiencies under 50% were utilized to ensurea multiplicity of infection of approximately 1. Pool composi-tion was measured at the beginning of the experiment, as wellas following recovery from an LD90 doxorubicin drug dose.Untreated cells that had grown in culture for the duration ofthe experiment were used as controls. Hairpin 97mersequences in the Mir30 context were as follows:

shBid-1- TGCTGTTGACAGTGAGCGCCACAGAAGATTC-CATATCAAATAGTGAAGCCACAGATGTATTTGATATG-GAATCTTCTGTGATGCCTACTGCCTCGGA,shBid-2- TGCTGTTGACAGTGAGCGCCACAGAAGATTC-CATATCAAATAGTGAAGCCACAGATGTATTTGATATG-GAATCTTCTGTGATGCCTACTGCCTCGGA.shCaspase 8- TGCTGTTGACAGTGAGCGAAACTATGAC-GTGAGCAATAAATAGTGAAGCCACAGATGTATTTATTG-CTCACGTCATAGTTCTGCCTACTGCCTCGGA

Background distribution analysis for in vivo screeningdata

A total of 454 sequencing data used to determine coefficientof variation (CV) thresholds is available from the GEO data-base (accession number GSE16090). CVs were calculated bydividing the SD of the fold change in read number (for anyhairpin with >4 reads) by the mean of the fold change. To beincluded in this analysis, hairpins had to be present at highenough quantities to have at least 4 reads. Previously validatedhit CVs (13) included IL-6, Lyn, Rac2, Twf, and CrkL and werecompared with the background distribution by t test. Cumu-lative distributions were plotted in Matlab by using thedfittool.

In Vivo shRNA Screen for Mediators of Therapeutic Response

www.aacrjournals.org Cancer Res; 71(17) September 1, 2011 5851




In vivo screeningSix-week-old C57BL/6J mice (Jackson Laboratories) were

tail-vein injected with 2 million Em-Myc p19ARF�/� lymphomacells expressing the 22 shRNAs targeting the Bcl-2 family.Infection was optimized such that each tumor cell expressed asingle shRNA. We sampled pool composition before injection,upon the development of a palpable tumor burden and uponrelapse following IP injection of 8 mg/kg of doxorubicin. For invivo screen validation, pure populations of cells expressingshRNAs were isolated by GFP sorting by using a fluorescence-activated cell sorting ARIA cell sorter. Two million cellstransduced with a vector control or specific shRNAs wereinjected into syngeneic recipient mice. At the presentation ofpalpable lymphomas, mice were treated with 10 mg/kg dox-orubicin and disease-free survival/tumor-free progressionwere monitored.

Screening analysisLuminex intensities for serial dilutions of PCR samples from

pooled hairpin libraries were compared by calculating a curvefit for a given sample by using the equation [Y ¼ a*(1�exp(�b*x)]. Sample integrals were then calculated, and theseintegral scores were compared after subtracting the initialfrom the final integral score. All independent Luminex runswere normalized to the maximum signal intensity and all runsdone on different days contained internal standards for day-to-day normalization. Scoring shRNAs were identified follow-ing a 3-step process. First, to eliminate a systematic error fordepleting hairpins, we transformed all the data by adding thehairpin mean to all in vivo measurements. We then applied 2filters. The first filter was based upon a comparison of thedistribution of a given shRNA with the variation of neutralhairpins. We required our "hits" to have a CV that was equal toor less than 0.4. This allowed us to threshold out approxi-mately 80% of neutral hairpins. The second filter was basedupon a comparison of treated samples with untreated hairpinsamples. To progress to further validation efforts, we requiredour treated sample to be different at the 0.10 significance levelversus untreated controls.

Data analysisLinear fits were calculated by using a least squares algo-

rithm. Sequences were compared for overlap by using the localalignment method of Smith–Waterman in the Matlab func-tion localalign.m. Heat maps were generated in Matlab.Kaplan–Meier analyses were carried out by using GraphPadPrism software.

Results

A bead-based assay for the direct measurement ofpooled shRNA representation

The Bcl-2 family consists of 16 pro- and 6 antiapoptoticproteins that regulate programmed cell death in response to adiverse set of intrinsic and extrinsic death stimuli (21, 22). Toassess how these genes modulate chemotherapy-induced celldeath across multiple in vivo contexts, as well as across diversein vitro conditions in a multiplexed manner, we adapted a

validated set of shRNAs targeting all 22 Bcl-2 family membersto Luminex bead–based analysis (Fig. 1A; ref. 18). This tech-nology has previously been used to quantify diverse sets ofmicroRNAs in solution with improved accuracy relative toclassic microarray approaches (23). Thus, we reasoned thatthis approach could be modified to carry out reproduciblequantification of subgenome-sized shRNA pools.

Briefly, we covalently coupled an amino-modified oligonu-cleotide that is the reverse complement of the unique guidestrand section of each shRNA to fluorescently labeled Luminexbeads. Our PCR strategy uses a forward primer complemen-tary to the microRNA loop sequence present in all hairpinsand a biotinylated reverse primer complementary to theflanking microRNA sequence (Fig. 1B). Thus, all shRNA guidestrands can be amplified by using common primers, and thequantity of individual shRNAs can be visualized with strepta-vidin–PE after bead hybridization. Notably, this approach isdistinct from barcoded shRNA libraries, in which shRNAs areidentifiable by a flanking DNA sequence. In this case, we usedthe unique portion of the shRNA, itself, as the barcode. Thisallows for multiplexed shRNA quantification in any vectorbackbone.

As an initial proof-of-principle experiment,we confirmed thateach Bcl-2 shRNA could be quantified by Luminex bead hybri-dization when starting with similar concentrations of dsPCRproduct (Supplementary Fig. S2A). Importantly, whereas eachPCR product with a cognate bead was readily detectable, acontrol probe exhibited no significant signal with any of the 22shRNAs in the plasmid library. In fact, the average maximumsignal for an shRNA present in the pool was approximately 100-fold higher than the control probe signal. Although this negativecontrol ruledout any largemagnitudenonspecific hybridization,wewanted to rule out smaller amounts of cross hybridization asthe source of the variation in the maximum fluorescenceintensity of the various probes (Supplementary Fig. S2B). Tothis end, we noted a strong relationship between probe GCcontent andmaximumsignal intensity. Oligonucleotides deviat-ing from this relationship were analyzed for local sequencealignments across the entire shRNA libraryutilizing thedynamicprogramming method of Smith–Waterman (24). The variationin the average local alignment bit scores for all "outlier" probeswas highly similar (Supplementary Fig. S2C), indicating thatcross-hybridization is an unlikely contributor to overall signalintensity. Thus, oligonucleotide sequences chosen for optimalsiRNA performance are well suited for hybridization-basedsequence identification.

We next carried out mock enrichment experiments inwhich known concentrations of genomic DNA from singlehairpin infected Em-Myc p19Arf�/� lymphoma cells were com-bined at distinct ratios (Supplementary Fig. S2D). Theselymphoma cells were derived from a well-established precli-nical mouse model of Burkitt's lymphoma and represent atractable setting to investigate the genetics of therapeuticresponse (25, 26). By using these cells, we observed a linear andhighly reproducible change in measured fluorescence inten-sity that tightly correlated with the known fold enrichment ofthe control sample across 8 fold changes in relative DNAabundance.

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Cancer Res; 71(17) September 1, 2011 Cancer Research5852




The Bcl-2 family differentially modulates therapeuticresponse in distinct B-cell tumorsThe initial validation of our measurement technology led us

to benchmark this approach against an established singleshRNA flow cytometry–based assay. In this assay, GFP wasused as a surrogate marker for the presence of each of 16distinct Bcl-2 family member shRNAs, and the impact of genesuppression was determined by the relative change in thepercent of GFP-positive cells following treatment (18). In eachcase, we examined the effect of Bcl-2 family gene knockdownon the in vitro response of Em-Myc p19Arf�/� lymphoma cells tothe front-line chemotherapeutic doxorubicin (Fig. 1C). Alinear relationship (r2 ¼ 0.89) was observed between multi-plexed bead-based measurements and single shRNA flowcytometry measurements of shRNA enrichment and depletionfollowing doxorubicin treatment. Thus, a bead hybridizationassay can rapidly and accurately measure shRNA pool com-position following drug selection.A key advantage of shRNA pool–based approaches lies in

their inherent adaptability to diverse experimental systemsand conditions. To test the flexibility of our system, weexamined the effects of Bcl-2 family member suppression

on doxorubicin response in a distinct cell line. In this case,we examined cells derived from a BCR-Abl–driven murinemodel of B cell acute lymphoblastic leukemia (B-ALL; ref. 27).As observed in the Burkitt's lymphoma model, we couldidentify a robust Bcl-2 family shRNA drug resistance andsensitivity profile in these cells. However, the shRNA signa-tures were distinct between cell types. The most obviousfeature differentiating the 2 cell lines was the critical rolefor the BH3-only member Bim in doxorubicin-induced celldeath in B-ALL (Fig. 1D). Because Bim levels are known toincrease in response to environmental but not genotoxicstress, the involvement of Bim in the response to a DNA-damaging agent in this context was unexpected. To explorethe mechanism of Bim-induced cell death, we examined Bimlevels in p185þ BCR-Abl ALL cells treated with genotoxicagents (doxorubicin and chlorambucil) and a histone deace-tylase inhibitor known to promote significant Bim induction(SAHA) in B cell malignancies (28). Notably, protein levels ofBim were induced acutely following treatment with DNA-damaging agents in B-ALL cells (Fig. 1E). These data highlightthe potential of pool-based shRNA approaches to identifytumor cell–specific determinants of therapeutic response.

Figure 1. Examining the role of the entire Bcl-2 family in therapeutic response. A, a schematic depicting the difference between single and pool-basedevaluation of shRNA composition. B, a diagram illustrating the Luminex-based shRNA PCR strategy. PCR primers were designed from constant regionsflanking all hairpins. After PCR amplification, a biotinylated primer is used to measure hairpin abundance, and the unique fluorescence of the Luminex beaddistinguishes hairpin identity. C, a comparison of the bead-based quantification of hairpin representation following doxorubicin treatment with single-cell flowcytometry measurements (18). Left, bead-based measurements are plotted against each corresponding single-hairpin measurement. Right, heat mapscomparing shRNA enrichment and depletion by using bead-based and flow cytometry approaches. D, a heat map comparing the impact of depletingeach Bcl-2 family hairpin on the response to doxorubicin treatment in Em-MYC p19Arf�/� lymphomas and p185 BCR-Ablþ p19Arf�/� B-ALLs. The asteriskdemarcates the differential impact of suppressing the BH3-only protein Bim on doxorubicin sensitivity in these 2 cell types. E, p185 BCR-Ablþ p19Arf�/� cellswere treated for 12 hours at an LD90 of the indicated compounds and analyzed for Bim levels by Western blot.






An in vivo screen for microenvironment-specificmodifiers of therapeutic response

A central challenge in the development of effective antic-ancer approaches is to understand the impact of the tumormicroenvironment on therapeutic response. To test whetherour system could be used to examine cancer therapy in vivo,we conducted a screen to identify Bcl-2 family members thatmodulate the response of lymphomas to doxorubicin. Here, allBcl-2 family shRNAs were simultaneously cotransfected intoviral packaging cells to produce a multiconstruct viral pool(Fig. 2A). The resulting pool was used to infect primary Em-Mycp19Arf�/� lymphoma cells ex vivo, and transduced cells weretail-vein injected into syngeneic recipient mice. A cohort of 8mice was sacrificed following tumor onset, and a secondcohort was treated with 8 mg/kg doxorubicin. Followingtumor relapse, lymphoma cells were harvested from lymphnodes and the thymus, 2 common sites of lymphoma man-ifestation in the Em-Myc mouse (25), and the relative shRNAcontent was compared between untreated and treated tumorsin these distinct tumor microenvironments.

A striking feature of the resulting data was the mouse-to-mouse variability in hairpin composition following drug treat-

ment. This suggests that the complexity of the in vivo micro-environment can substantially influence the measured effectof a relatively neutral shRNA. Further inspection of thisvariation suggested that a subset of shRNAs exhibited a levelof variation comparable with the in vitro data, whereas theremainder showed significantly higher fluctuation (Fig. 2B). Ifotherwise neutral hairpins exhibit larger variation in in vivodatasets, the size of this variation may represent a meaningfuldiscriminator to focus on hairpins whose effects are large andreproducible enough to overcome this variability. To deter-mine whether such variation is a consistent feature of in vivodatasets, we made use of a comprehensive in vivo versus invitro shRNA screening dataset (13). Indeed, most shRNAsexhibited highmouse-to-mouse CVs in vivo (Fig. 2C). However,when we focused on shRNAs shown to exert a biological effectin subsequent validation experiments, we saw a significantdecrease in shRNA CVs (P < 0.01). Thus, variation present inthis established dataset can be used to generate a CV thresh-old that identifies shRNAs with a high probability of exerting arelevant biological effect. This cutoff was then employed tofilter data generated by using our Luminex approach (Fig. 2D).As a test of the relevance of this variation cutoff to other drug

Figure 2.An in vivo shRNA screen for modulators of doxorubicin response. A, a diagram depicting the in vivo screening strategy. Pooled shRNAs targeting Bcl-2 family members were retrovirally transduced into Em-MYC p19Arf�/� lymphomas. shRNA composition was measured either following the presentation ofpalpable tumor burden or following tumor relapse after treatment with 8 mg/kg doxorubicin. B, a comparison of the CV for hairpins targeting the Bcl-2 family incell culture versus in vivo screens by using a Luminex measurement methodology. C, an analysis of the distribution of shRNA CV values for a large in vitroversus in vivo screening dataset generated by high-throughput sequencing. The vertical line represents the CV threshold used to filter in vivo data. The arrowsdenote averages for validated hairpins relative to all hairpins. D, Bcl-2 family "hits" following filtering for CV and enrichment criteria and separated byanatomical niche. The asterisk denotes a Bcl-w shRNA that scored as enriched in the thymus but was excluded because of the lack of Bcl-w expression in thiscontext.

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screens, we examined the stochastic variation in the repre-sentation of vector control infected Em-Myc tumor cells fol-lowing treatment with the microtubule poison vincristine invivo. Here, we transplanted tumor cells into recipient mice at adefined infection efficiency, as monitored by GFP expression,and examined the variation in the percentage of GFP-positivecells in distinct mice following treatment. Importantly, thesecontrols exhibited an in vivo CV that was greater than thedoxorubicin variation cutoff (Supplementary Fig. S3A and B),suggesting that the CV threshold established in this study maybe broadly applicable to other datasets.As additional criteria for examining in vivo screening data,

we required that shRNA target mRNAs be present in untrans-duced lymphoma cells and that the representation of a"scoring" shRNA be significantly enriched or depleted as aconsequence of doxorubicin treatment (see methods). Theresulting list of scoring shRNAs included Bcl-2 family mem-bers previously described to influence therapeutic response(ref. 17; Fig. 2D and Supplementary Table S1). For instance, wefound that suppression of the BH3-only protein Puma pro-moted doxorubicin resistance in both the lymph node andthymus compartments, consistent with previous reportsexamining either B lymphoma cells or thymocytes (26, 29).Thus, this approach can readily identify important regulatorsof drug-induced cell death.

The extrinsic death pathway is a thymus-specificmediator of therapeutic responseInterestingly, in contrast with Puma and other general cell

death regulators, we identified the proapoptotic Bcl-2 familymember Bid as a specific mediator of doxorubicin cytotoxicityin the thymus but not in the lymph nodes. To validate andextend the genetic result in light of this finding, we conductedan in vivo GFP competition assay in the spleen, bone marrow,peripheral lymph nodes, and thymus. In this assay, GFPpositivity is used as a surrogate marker for the presence ofa Bid shRNA, and the impact of Bid suppression is determinedby the relative change in the percent of GFP-positive tumor

cells. Consistent with the initial screening data, Bid lossimpaired lymphoma cell death in the thymic tumor micro-environment but not other tumor microenvironments (Fig. 3Aand B). This tissue specificity was not because of the selectiveexpression of these proteins in specific tumor microenviron-ments, as we observed similar levels of Bid and its upstreamregulator caspase 8 in the tumor-bearing lymph node andthymus (Supplementary Fig. S4A and B). Notably, Bid is uniqueamong Bcl-2 family members in that it translocates to themitochondria following extrinsic activation of death receptors(30, 31). Thus, these data are consistent with a mechanismwhereby constitutively present Bid is activated following therelease of secreted factors or tumor–stromal cell interactionsthat are specific to the treated thymic microenvironment.

The relevance of Bid to DNA damage–induced deathremains a subject of debate (32, 33). To confirm the impor-tance of Bid to doxorubicin-induced cell death in vivo, weinjected 3 cohorts of syngeneic mice with Em-Myc p19Arf�/�

lymphoma cells expressing 1 of 2 validated shRNAs targetingBid or a vector control. At tumor onset, all mice were treatedwith 10 mg/kg doxorubicin and monitored for tumor regres-sion and relapse. Suppression of Bid resulted in decreasedtumor-free survival and tumor cell clearance compared withcontrol tumors (Fig. 4A). Furthermore, in mice-bearing shBid-transduced lymphomas, 50% of the mice showed no tumor-free survival, whereas 90% of control mice exhibited a periodof tumor-free survival. Notably, in the case of the lymphomacells used in this study, suppression of Bid in vitro hadminimaleffect on lymphoma cell survival following doxorubicin treat-ment (Fig. 4B). Thus, treatment of these tumors in their nativemicroenvironment reveals genetic dependencies that are notpresent in cultured cells.

These data suggest that activation of components of theextrinsic cell death pathway potentiate chemotherapeutic effi-cacy in the thymus. To further interrogate the role of deathreceptor signaling in therapeutic response in this setting, wetargeted caspase 8, the direct activator of Bid by generatinghairpins targeting caspase 8 (Supplementary Fig. S4C).

Figure 3. Bid potentiates doxorubicin efficacy in the thymus. A, a graph depicting an in vivo GFP competition assay in lymphoma cells partially transducedwith shBid-1. Mice were injected with partially transduced lymphoma populations. At tumor onset, all mice were treated with 10 mg/kg doxorubicin.Seventy-two hours after treatment, mice were sacrificed and surviving lymphoma populations were harvested. Fold change in GFP percentage wasassessed 48 hours later (n � 4). B, hematoxylin and eosin–stained sections from mice-bearing vector control or shBid thymic lymphomas. Mice weretreated with 10 mg/kg doxorubicin and sacrificed 48 hours later. Representative fields from treated and untreated are shown at 25� magnification.Dark patches in treated vector control tumors indicate sites of normal lymphocyte infiltration.






Suppression of caspase 8 in transplanted lymphomas phenocop-ied the effect of Bid silencing asmeasured by tumor-free survival(Fig. 5A), suggesting an upstream induction of death receptorsignaling in the thymus following doxorubicin treatment.

Finally, to confirm the specificity of Bid-induced cell deathin the thymus relative to whole organism chemotherapeuticresponse, we examined the effect of Bid suppression ontumor-free survival following doxorubicin treatment in athy-mic mice. Pure populations of either shBid or vector controltransduced lymphoma cells were transplanted into surgicallythymectomized recipient mice. Upon the presentation of apalpable disease burden, mice were dosed with 10 mg/kg ofdoxorubicin and monitored for tumor-free and overall survi-val. In this context, chemotherapeutic response was indis-tinguishable in the presence or absence of Bid (Fig. 5B).

Discussion

We have presented a tractable methodology for pooledshRNA screens that can be rapidly adapted to diverse vectorsystems and gene families. The value of this system is exem-plified by the rapid manner in which drug function can beinterrogated in multiple cell types in vitro and in diverseanatomical contexts in vivo. Importantly, although numerouscell culture—based loss of function screens have been carriedout to identify modulators of therapeutic response, this is thefirst report to describe an in vivo loss of function therapyscreen-–the relevance of which is apparent in light of thediscordant in vitro and in vivo phenotypes resulting from Bidsuppression. Whereas the set of shRNAs probed in this work isrestricted to a particular aspect of cell biology, recentadvances in bead-based DNA hybridization now permit thesimultaneous resolution of as many as 500 distinct oligonu-cleotides so that multiple facets can be explored simulta-neously. We have recently shown that as many as 1,000distinct shRNAs can be introduced into individual mice(13), suggesting that large shRNA libraries can be combinedwith this technology to probe the impact of myriad geneticlesions in diverse pathophysiologic contexts.

In this study, we systematically examined shRNAs targetingthe entire Bcl-2 family in multiple in vitro and in vivo settings.In all therapeutic contexts, we identified a specific BH3"activator" gene essential for mediating the effects of front-line chemotherapy. Biochemical studies have previouslydefined critical roles for the BH3 only family members Bid,

Figure 4. Bid status affects therapeutic outcome in vivo, but not in vitro. A,a Kaplan–Meier curve showing tumor-free survival in mice-bearingvector or shBid lymphomas. All mice were treated with a single dose of 10mg/kg doxorubicin (n � 10). Inset, a Western blot showing Bid proteinlevels in the presence of Bid shRNAs. B, a dose–response curveshowing the relative viability of lymphoma cells treated with doxorubicinin vitro for 48 hours. Lymphoma cells were transduced with either avector control or an shRNA targeting Bid.

Figure 5. The extrinsic deathpathway mediates doxorubicinresponse in the thymus. A, aKaplan–Meier curve showingtumor-free survival of mice-bearing vector, shBid, orshCaspase 8–expressinglymphomas. All mice were treatedwith a single dose of 10 mg/kgdoxorubicin (n � 10). B, a Kaplan–Meier curve showing the tumor-free survival of thymectomizedmice transplanted with purepopulations of lymphoma cellstransduced with shBid or a vectorcontrol. All mice were treated witha single dose of 10 mg/kgdoxorubicin (n ¼ 6 for bothcohorts).

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Puma, and Bim. Furthermore, the recent development of a Bid,Puma, and Bim triple knockout mouse confirmed the essentialrole of these proteins in developmentally regulated apoptosis(34). Interestingly, our data suggests that the relevant "acti-vator" protein can vary quite significantly in neoplastic cells.Although an "activator" is always necessary for cell death, thecellular environment or driving oncogene can dramaticallyshift the precise BH3-only family member that is most relevantfor therapy-induced apoptosis. This context-dependent rele-vance of apoptotic regulators may underlie the significantchallenge in eradicating disseminated malignancies and high-lights the need to understand the relationship between intrin-sic and paracrine signals and Bcl-2 family regulation.An unexpected finding from this work is that although

caspase 8 and Bid are expressed at similar levels in diversetumor-bearing locations in vivo, they are specifically requiredfor drug efficacy in the thymus. Examined in isolation, thisresult would suggest that the thymus is a prodeath micro-environment. However, previous studies in thymectomizedmice have shown that the thymus can exert a net protectiveeffect on tumor cells following doxorubicin treatment (9). Thiscytoprotective effect is mediated in a paracrine fashion bythymic endothelial cells that secrete multiple prosurvivalcytokines in response to DNA damage. Cytokine inductionsubsequently upregulates Bcl-xL and promotes the survival oftarget tumor cells. Thus, the unique role of death receptoractivity in this context may function to counterbalanceunchecked survival signaling following cellular stress in the

thymic microenvironment. Notably, however, the precisemechanism of death receptor engagement in this contextremains to be determined. Addition of recombinant deathreceptor ligands, such as FASL, TNF, and TRAIL in vitro fails toinduce lymphoma cell death. Consequently, the engagementof death receptor signaling, like survival signaling, may requirea more complex concerted action of secreted factors and cell–cell or cell–stromal interactions.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank members of the Lauffenburger and Hemann labs forhelpful comments and criticisms. We would also like to thank Hai Jiang forimportant conceptual and technical contributions to this study.

Grant Support

M.T. Hemann is supported by NIH RO1 CA128803 and the Ludwig Founda-tion. J.R. Pritchard is a Poitras Graduate Fellow, and J.R. Pritchard and C.E.Meacham are supported by the MIT Department of Biology training grant. L.A.Gilbert is supported by a Ludwig Fellowship. Additional funding was providedby the Integrated Cancer Biology Program grant NCI 1-U54-CA112967 to D.A.Lauffenburger and M.T. Hemann.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 24, 2011; revised June 20, 2011; accepted July 15, 2011;published OnlineFirst July 22, 2011.

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2011;71:5850-5858. Published OnlineFirst July 22, 2011.Cancer Res Justin R. Pritchard, Luke A. Gilbert, Corbin E. Meacham, et al. Determinants of Chemotherapeutic ResponseBcl-2 Family Genetic Profiling Reveals Microenvironment-Specific

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