Reports

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Phthalimide-based drugs emerged in the 1950s. Among the most notable was thalidomide, developed initially as a sedative but infamously withdrawn from human use owing to catastrophic teratogenicity (1). More recently, the phthalimides have been successfully repurposed for erythema nodosum leprosum, multiple myeloma (MM) and myelodyspasia. The efficacy of thalidomide, lenalidomide and pomalidomide in MM (Fig. 1A), has prompted investigation into the mechanism-of-action of phthalimide immunomodulatory drugs (IMiDs). Using ligand-affinity chromatography Cereblon (CRBN), a component of a cullin-RING ubiquitin ligase (CRL) complex was identified as the target of thalidomide (2). Recently our group and others reported that phthalimides prompt CRBN-dependent proteasomal degradation of transcription factors IKZF1 and IKZF3 (3, 4). Crystallographic studies now establish that IMiDs bind CRBN to form a cryptic interface that promotes recruitment of IKZF1 and IKZF3 (5).

Ligand-induced target protein destabilization has proven to be an efficacious therapeutic strategy, in particular for cancer as illustrated by arsenic trioxide-mediated degrada-tion of PML in acute promyelocytic leukemia (6) and estro-gen receptor degradation by fulvestrant (7). Historically, target-degrading compounds have emerged serendipitously

or through target-specific cam-paigns in medicinal chemistry. Chemical biologists have devised elegant solutions to modulate protein stability using engi-neered cellular systems, but the-se approaches have been limited to non-endogenous fusion pro-teins (8–11). Others have achieved the degradation of en-dogenous proteins through the recruitment of E3 ligases, but these approaches have been lim-ited by the requirement of pep-tidic ligands (12–14), the use of non-specific inhibitors (15), and by low cellular potency (EC50 25 – 150 μM).

RING-domain E3 ubiquitin-protein ligases lack enzymatic activity, rather functioning as adaptors to E2 ubiquitin-conjugating enzymes. Inspired by the retrieval of CRBN using a tethered thalidomide (2), we hy-pothesized that rational design of bifunctional phthalimide-conjugated ligands could confer CRBN-dependent target protein degradation as chemical adapt-ers. We selected BRD4 as an ex-

emplary target. BRD4 is a transcriptional co-activator that binds to enhancer and promoter regions, via recognition of acetylated lysines on histone proteins and transcription fac-tors (TFs) (16). Recently, we developed a direct-acting inhib-itor of BET bromodomains (JQ1) (17), that displaces BRD4 from chromatin leading to impaired signal transduction from TFs to RNA Polymerase II (18–20). Silencing of BRD4 expression by RNA interference in murine and human mod-els of MM and acute myeloid leukemia (AML) elicited rapid transcriptional down-regulation of the MYC oncogene and a potent anti-proliferative response (19, 21). These and other studies in cancer, inflammation (22) and heart disease (23, 24), establish a desirable mechanistic and translational pur-pose to target BRD4 for selective degradation.

Having shown that the carboxyl group on JQ1 (25) and the aryl ring of thalidomide (5) can tolerate chemical substi-tution, we designed the bifunctional dBET1 to have pre-served BRD4 affinity and an inactive epimeric dBET1 (R) as a stereochemical control (Fig. 1, A and B). Selectivity profil-ing confirmed potent and BET-specific target engagement among 32 bromodomains (BromoScan) (Fig. 1C and tables S1 and S2). A high-resolution crystal structure (1.0 Å) of dBET1 bound to BRD4(1) confirmed the mode of molecular recognition, comparable to JQ1 (Fig. 1D, fig. S1, and table

Phthalimide conjugation as a strategy for in vivo target protein degradation Georg E. Winter,1* Dennis L. Buckley,1* Joshiawa Paulk,1 Justin M. Roberts,1 Amanda Souza,1 Sirano Dhe-Paganon,2 James E. Bradner,1,3† 1Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA. 2Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA. 3Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.

*These authors contributed equally to this research.

*Corresponding author. E-mail: james_bradner@dfci.harvard.edu

The development of effective pharmacological inhibitors of multidomain scaffold proteins, notably transcription factors, is a particularly challenging problem. In part, this is because many small-molecule antagonists disrupt the activity of only one domain in the target protein. We devised a chemical strategy that promotes ligand-dependent target protein degradation using as an example the transcriptional coactivator BRD4, a protein critical for cancer cell growth and survival. We appended a competitive antagonist of BET bromodomains with phthalimide-conjugates to hijack the Cereblon E3 ubiquitin ligase complex. The resultant compound, dBET1, induced highly selective Cereblon-dependent BET protein degradation in vitro and in vivo and delayed leukemia progression in mice. A second series of probes resulted in selective degradation of the cytosolic protein, FKBP12. This chemical strategy for controlling target protein stability may have implications for therapeutically targeting previously intractable proteins.

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S3). Using the dBET1-BRD4(1) crystal structure and the re-cently reported structure of CRBN bound to thalidomide (5), we have modeled the feasibility of ternary complex for-mation in silico. An extended conformation of dBET1 was capable of bridging ordered BRD4(1) and CRBN without destructive steric interactions (Fig. 1E). To experimentally assess the chemical adaptor function of dBET1, we estab-lished a homogeneous proximity assay for recombinant, human CRBN-DDB1 and BRD4(1) (Fig. 1F). Luminescence arising from proximity of CRBN-DDB1- and BRD4-bound acceptor-donor beads increases with low concentrations of dBET1, and decreases at higher concentrations consistent with saturation of CRBN and BRD4 binding sites by excess ligand. Competitive co-incubation with free JQ1 or thalido-mide inhibits ternary complex formation, in a stereo-specific manner (Fig. 1G).

To assess the effect of dBET1 in cells, we treated a hu-man AML cell line (MV4;11) for 18 hours with increasing concentrations of dBET1 and assayed endogenous BRD4 levels by immunoblot. Pronounced loss of BRD4 (> 85%) was observed with concentrations of dBET1 as low as 100 nM (Fig. 1H). The epimeric control dBET1(R) was inactive (Fig. 1B), demonstrating that BRD4 degradation requires target protein engagement (fig. S2, A and B). The kinetics of BRD4 degradation were next determined using 100 nM dBET1 in MV4;11 cells. Marked depletion of BRD4 was ob-served at 1 hour and complete degradation was observed at 2 hours of treatment (Fig. 1I). A partial recovery in BRD4 abundance at 24 hours establishes the possibility of com-pound instability, a recognized liability of phthalimides. To quantify dose-responsive effects on BRD4 protein stability, we developed a cell-count normalized, high-content assay using adherent SUM149 breast cancer cells (Fig. 1J). Deple-tion of BRD4 was observed for dBET1 (EC50 = 430 nM) without apparent activity for dBET1(R), confirmed by im-munoblot (fig. S2, C and D). Additional cultured adherent and non-adherent human cancer cell lines showed compa-rable response (SUM159, MOLM13) (fig. S3).

To explore the mechanism of dBET1-induced BRD4 deg-radation, we studied requirements on proteasome function, BRD4 binding and CRBN binding, using chemical genetic and gene-editing approaches. First, we confirmed that treatment with either JQ1 or thalidomide alone was insuffi-cient to induce BRD4 degradation in MV4;11 cells (fig. S4A). Degradation of BRD4 by dBET1 was rescued by the pro-teasome inhibitor carfilzomib, establishing a requirement for proteasome function (Fig. 2A). Pre-treatment with excess JQ1 or thalidomide abolished dBET1-induced BRD4 degra-dation, consistent with a requirement for both BRD4 and CRBN engagement (Fig. 2A). Pre-treatment with the NAE1 inhibitor MLN4924 (26) rescued BRD4 stability establishing dependence on CRL activity, as expected for cullin-based ubiquitin ligases that require neddylation for processive E3 ligase activity (4, 27) (fig. S4B). Finally, to definitively con-firm the cellular requirement for CRBN, we used a previous-

ly published CRBN deficient human MM cell line (MM1.S-CRBN−/−) (4). Whereas treatment of MM1.SWT cells with dBET1 promoted degradation of BRD4, exposure of MM1.S-CRBN−/− cells to dBET1 was ineffectual (Fig. 2B). These data provide mechanistic evidence for CRBN-dependent pro-teasomal degradation of BRD4 by dBET1.

To assess the feasibility of extending this strategy to oth-er protein targets, we designed and synthesized phthalimide-conjugated ligands to the cytosolic signaling protein, FKBP12 (FKBP1A). FKBP12 has been extensively studied in the chemical biology literature, including studies of engineered target degradation and a growing literature on chemical dimerizers. At a known permissive site on the FKBP12-directed ligand SLF, we positioned two chemical spacers to create the conjugated phthalimides dFKBP-1 and dFKBP-2 (Fig. 2C). Both potently decreased FKBP12 abun-dance in MV4;11 cells (Fig. 2D). As with dBET1, destabiliza-tion of FKBP12 by dFKBP-1 was rescued by pre-treatment with carfilzomib, MLN4924, free SLF or free thalidomide (Fig. 2E). CRBN-dependent degradation was established using previously published isogenic 293FT cell lines which are wild-type (293FT-WT) or deficient (293FT-CRBN−/−) (4) for CRBN (Fig. 2F).

An unbiased, proteome-wide approach was selected to assess the cellular consequences of dBET1 treatment on pro-tein abundance, in a quantitative and highly parallel format (28). We compared the immediate impact of dBET1 treat-ment (250 nM) to JQ1 and vehicle controls in MV4;11 cells. A two hour incubation was selected to capture primary, immediate consequences of small-molecule action and to mitigate expected, confounding effects on suppressed trans-activation of BRD4 target genes. Three biological sample replicates were prepared for each treatment condition using isobaric tagging that allowed the detection of 7,429 proteins. Following JQ1 treatment, few proteomic changes are ob-served (Fig. 3A and table S4). Only MYC was significantly depleted by more than 2-fold after 2 hours of JQ1 treatment, confirming the reported rapid and selective effect of BET bromodomain inhibition on MYC expression in AML (Fig. 3, A and C) (21). JQ1 treatment also down-regulated the onco-protein PIM1 (Fig. 3, A and C).

Treatment with dBET1 elicited a comparable, modest ef-fect on MYC and PIM1 expression. Remarkably, only three additional proteins were identified as significantly (p < 0.001) and markedly (> 5-fold) depleted in dBET1-treated cells: BRD2, BRD3 and BRD4 (Fig. 3, B and C). These find-ings are consistent with the anticipated, BET-specific bro-modomain target spectrum of the JQ1 bromodomain-biasing element on dBET1 (17). The remaining BET-family member BRDT is not detectable in MV4;11 cells. To validate these findings, we measured BRD2, BRD3, BRD4, MYC and PIM1 levels by immunoblot following compound treatment, as above. BET family members were degraded only by dBET1, whereas MYC and PIM1 abundance was decreased by both dBET1 and JQ1, and to a lesser degree (Fig. 3D). No effect on

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Ikaros TF expression was observed in either treatment con-dition (fig. S5). MYC and PIM1 are often associated with massive adjacent enhancer loci by epigenomic profiling, (18, 20) suggestive of a transcriptional mechanism of down-regulation. We therefore measured mRNA transcript abun-dance for each depleted gene product (Fig. 3E). Treatment with either JQ1 or dBET1 down-regulated MYC and PIM1 transcription, suggestive of secondary transcriptional ef-fects. Transcription of BRD4 and BRD3 were unaffected, consistent with post-transcriptional effects. Interestingly, transcription of BRD2 was affected by JQ1 and dBET1, whereas protein stability of the BRD2 gene product was on-ly influenced by dBET1, suggestive of transcriptional and post-transcriptional consequences. These data establish a highly selective effect of dBET1 on target protein stability, proteome-wide.

We next explored the differential anti-proliferative con-sequences of BET degradation with dBET1 to BET bromo-domain inhibition with JQ1. Degradation of BRD4 by dBET1 was associated with an enhanced apoptotic response in MV4;11 AML and DHL4 lymphoma cells, as measured by caspase activation (Caspase-GLO) (Fig. 4A), cleavage of poly(ADP-ribose) polymerase (PARP), cleavage of Caspase-3 (immunoblot) (Fig. 4B) and Annexin V staining (flow cy-tometry) (fig. S6A). Kinetic studies revealed an apoptotic advantage for dBET1 after pulsed treatment followed by washout in MV4;11 cells (Fig. 4, C and D). Indeed, dBET1 induced a potent and superior inhibitory effect on MV4;11 cell proliferation at 24 hours (Fig. 4E).

The rapid biochemical activity and robust apoptotic re-sponse of cultured cell lines to dBET1 established the feasi-bility of assessing effects on primary human AML cells, where ex vivo proliferation is negligible in short-term cul-tures. Exposure of primary leukemic patient blasts to dBET1 elicited dose-proportionate depletion of BRD4 (Fig. 4F) and improved apoptotic response compared to JQ1 (Annexin V/PI staining) (Fig. 4G and fig. S6B). Together, these data exemplify that target degradation can elicit a more pro-nounced biological consequence than domain-specific target inhibition.

To model the therapeutic opportunity of BRD4 degrada-tion in vivo, we first evaluated the tolerability and anti-tumor efficacy of dBET1 in a murine hind limb xenograft model of human MV4;11 leukemia cells (29). As pharmaco-kinetic studies of dBET1 indicated adequate drug exposure in vivo (fig. S7A), tumor-bearing mice were treated with dBET1 administered by intraperitoneal injection (50 mg/kg daily) or vehicle control. After 14 days of therapy a first tu-mor reached institutional limits for tumor size, and the study was terminated for comparative assessment of efficacy and modulation of BRD4 stability and function. Administra-tion of dBET1 attenuated tumor progression as determined by serial volumetric measurement (Fig. 4H), and decreased tumor weight assessed post-mortem (fig. S8A). Acute phar-macodynamic degradation of BRD4 was observed 4 hours

after a first or second daily treatment with dBET1 (50 mg/kg IP) by immunoblot (Fig. 4I), accompanied by down-regulation of MYC (Fig. 4I). These findings were confirmed by immunohistochemistry at the conclusion of the 14 day efficacy study (Fig. 4J). A statistically significant destabiliza-tion of BRD4, down-regulation of MYC and inhibition of proliferation (Ki67 staining) was observed with dBET1 com-pared to vehicle control in excised tumors (Fig. 4J and fig. S8B). Notably, two weeks of dBET1 was well tolerated with preservation of animal weight and normal complete blood counts (fig. S7, C and D). To compare the efficacy of BET inhibition to BET degradation, we selected an aggressive disseminated leukemia model (mCherry+ MV4;11) and treat-ed animals with established disease using equimolar con-centrations of JQ1 and dBET1 for 19 days. Post-mortem analysis of leukemic burden in bone marrow by FACS re-vealed significantly decreased mCherry+ disease with dBET1 administration (Fig. 4K).

In summary, we present a mechanism-based, chemical strategy for endogenous target protein degradation. Phthalimide conjugation to selective small molecules pro-duces CRBN-dependent post-translational degradation with exquisite target-specific activity. Though this approach is CRBN-dependent, CRBN is ubiquitously expressed in physi-ologic and pathophysiologic tissues, supporting broad utility in developmental and disease biology. The increased apop-totic response of primary AML cells to dBET1, even com-pared to the efficacious tool compound JQ1, highlights the potential superiority of BET degradation over BET bromo-domain inhibition and prompts consideration of therapeutic development. Pharmacologic destabilization of BRD4 in vivo also resulted in improved anti-tumor efficacy in a human leukemia xenograft compared to JQ1. The extension of this approach to new targets (here, FKBP12), low molecular mass, high cell permeability, potent cellular activity, and synthetic simplicity addresses limitations associated with prior, pioneering systems. We anticipate dFKBP1 will also serve as a useful tool for the research community, in control of fusion protein stability. A more general implication of this research is the feasibility of approaching intractable protein targets using phthalimide-conjugation of target-binding ligands that may or may not possess target-specific inhibitory activity.

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ACKNOWLEDGMENTS

We thank W. Kaelin, S. Orkin, and R. Mazitschek for engaging discussions and cellular reagents (WK); S.-H. Seo and S. Deangelo for assistance in the purification of BRD4; C. Ott for help with in vivo model studies; and N. Thoma for CRBN expression reagents. This research was supported by generous philanthropic gifts from Marc Cohen and Alain Cohen, the William Lawrence and Blanche Hughes Foundation, and the NIH (R01-CA176745 and P01-CA066996 to J.E.B.). G.E.W. is supported by an EMBO long-term fellowship. D.L.B. is a Merck Fellow of the Damon Runyon Cancer Research Foundation (DRG-2196-14). Atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession code 4ZC9). Quantitative proteomics studies were performed by R. Kunz of the Thermo Fisher Scientific Center for Multiplexed Proteomics at Harvard Medical School. Dana-Farber Cancer Institute has filed patent applications (62/096,318; 62/128,457; 62/149,170) that include the dBET and dFKBP compositions described in this manuscript. J.E.B. is a Founder of Tensha Therapeutics, a biotechnology company that develops drug-like derivatives of JQ1 as investigational cancer therapies.

SUPPLEMENTARY MATERIALS www.sciencemag.org/content/348/6237/aab1433/suppl/DC1 Materials and Methods Figs. S1 to S8 Tables S1 to S3 References (30–36) 17 March 2015; accepted 06 May 2015 Published online 21 May 2015 10.1126/science.aab1433

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Fig. 1. Design and characterization of dBET1. (A) Chemical structure of JQ1(S), phthalimides and dBET1. (B) Vehicle-normalized BRD4 displacement by AlphaScreen (triplicate means ± SD). (C) Selective displacement of phage-displayed BETs by dBET1 (BromoScan at 1 μM; n = 1). (D) Crystal structure of dBET1 bound to BRD4 bromodomain 1 (E) Docking of (D) into the published DDB1-CRBN structure (F) dBET1-induced ternary complex formation of recombinant BRD4(1) and CRBN-DDB1, by AlphaScreen (quadruplicate means ± SD; normalized to DMSO). (G) Competition of dBET1 (111 nM) induced proximity as in (F) in the presence of vehicle (DMSO), JQ1, thal-(–), JQ1(R) and thal-(+) all at 1 μM. Values represent quadruplicate means ± SD, normalized to DMSO. (H) Immunoblot for BRD4 and vinculin after 18 hours of treatment of MV4;11 cells with the indicated concentrations of dBET1. (I) Immunoblot for BRD4 and vinculin after treatment of MV4;11 cells with 100 nM dBET1 for the indicated exposures. (J) Cell count normalized BRD4 levels as determined by high-content imaging in SUM149 cells treated with the indicated concentrations of dBET1 and dBET1(R) for 18 hours. Values represent triplicate means ± SD, normalized to DMSO treated cells and baseline-corrected using immunoblots found in fig. S2C.

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Fig. 2. Chemical and genetic rescue of dBET1 and dFKBP-1 mediated degradation. (A) Immunoblot for BRD4 and vinculin after a 4-hour pre-treatment with either DMSO, Carfilzomib (400 nM), JQ1 (10 μM) or thalidomide (10 μM) followed by a 2-hour dBET1 treatment (100 nM) in MV4;11 cells. (B) Immunoblot for BRD4, CRBN and tubulin after treatment of MM1SWT or MM1SCRBN−/− with dBET1 for 18 hours at the indicated concentrations. (C) Structures of dFKBP-1 and dFKBP-2. (D) Immunoblot for FKBP12 and vinculin after 18 hours of treatment with the indicated compounds. (E) Immunoblot for FKBP12 and vinculin after a 4-hour pre-treatment with either DMSO, Carfilzomib (400 nM), MLN4924 (1 μM), SLF (20 μM) or thalidomide (10 μM) followed by a 4-hour dFKBP-1 treatment (1 μM) in MV4;11 cells. (F) Immunoblot for FKBP12, CRBN and tubulin after treatment of 293FTWT or 293FTCRBN−/− with dFKBP12 at the indicated concentrations for 18 hours.

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Fig. 3. Selective BET bromodomain degradation established by expression proteomics. MV4;11 cells were treated for 2 hours with DMSO, 250 nM dBET1 or 250 nM JQ1. (A) Scatterplat of fold-change in abundance of 7429 proteins comparing JQ1 to vehicle (DMSO) treatment, versus p-value (t-test; triplicate analysis). (B) As for (A) but comparing 250 nM dBET1 to vehicle treatment. (C) Bar graph of selected proteins from (A) and (B) normalized to vehicle. Values represent triplicate means ± SD. (D) Immunoblot of BRD2, BRD3, BRD4, MYC, PIM1 and VINC after a 2-hour treatment of MV4;11 cells with DMSO, 250 nM dBET1 or 250 nM JQ1. (E) qRT-PCR analysis of transcript levels of BRD2, BRD3, BRD4, MYC and PIM1 after a 2-hour treatment of MV4;11 cells with DMSO, 250 nM dBET1 or 250 nM JQ1. Values represent triplicate means ± SD.

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Fig. 4. The kinetic and anti-leukemic advantage of BET bromodomain degradation. (A) Bar graph depiction of fold increase of apoptosis assessed via Caspase-Glo assay relative to DMSO treated controls, 24 hours of treatment in MV4;11 or DHL4 cells. Values represent quadruplicate means ± SD. (B) Immunoblot for cleaved caspase 3, PARP cleavage and vinculin after treatment with dBET1 and JQ1 for 24 hours, as indicated. (C) Bar graph depiction of fold increase of apoptosis (Caspase-Glo) relative to DMSO treated controls. MV4;11 cells were treated for 4 or 8 hours with JQ1 or dBET1 at the indicated concentrations. Drug was washed out with PBS (3x) before being plated in drug-free media for a final treatment duration of 24 hours. Values represent quadruplicate means ± SD. (D) Immunoblot for cleaved caspase 3, PARP cleavage and vinculin after treatment conditions as described in (C). (E) Dose-proportional effect of dBET1 and JQ1 (24 hours) on MV4;11 cellular viability as approximated by ATP-dependent luminescence. Values represent quadruplicate means ± SD. (F) Immunoblot for BRD4 and vinculin after treatment of primary patient cells with the indicated concentrations of dBET1 for 24 hours. (G) Annexin V positive primary patient cells after 24 hours of treatment with either dBET1 or JQ1 at the indicated concentrations. Values represent the average of duplicates and the range as error bars (representative scatter plots in fig. S6). (H) Bar graph depiction (means ± SEM) of tumor volume of vehicle treated mice (n = 5) or mice treated with dBET1 (50 mg/kg; n = 6) for 14 days. (I) Immunoblot for BRD4, MYC and vinculin using tumor lysates from mice treated either once for 4 hours or twice for 22 hours and 4 hours, compared to a vehicle treated control. (J) Immunohistochemistry for BRD4, MYC and Ki67 of a representative tumor of a dBET1-treated and a control-treated mouse (quantification of 3 independent areas in fig. S8). (K) Bar graph (means ± SEM) of percent mCherry+ leukemic cells in flushed bone marrow from disseminated MV4;11 xenografts after daily treatment with dBET1 (n = 8) and JQ1 (n = 8) (both at 63.8 μmol/kg), or formulation control (n = 7) for 19 days.

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Phthalimide conjugation as a strategy for in vivo target protein degradation

BradnerGeorg E. Winter, Dennis L. Buckley, Joshiawa Paulk, Justin M. Roberts, Amanda Souza, Sirano Dhe-Paganon and James E.

published online May 21, 2015

ARTICLE TOOLS http://science.sciencemag.org/content/early/2015/05/20/science.aab1433

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2015/05/20/science.aab1433.DC1

REFERENCES

http://science.sciencemag.org/content/early/2015/05/20/science.aab1433#BIBLThis article cites 36 articles, 8 of which you can access for free

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Copyright © 2015, American Association for the Advancement of Science

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