Phthalimide conjugation as a strategy for in vivo target protein degradation by Georg E. Winter, Dennis L. Buckley, Joshiawa Paulk, Justin M. Roberts, Amanda Souza, Sirano Dhe-Paganon, and James E. Bradner Science Volume 348(6241):1376-1381 June 19, 2015 Published by AAAS

Fig. 1 Design and characterization of dBET1. 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 dimethyl sulfoxide (DMSO)]. (G) Competition of 111 nM dBET1-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 (VINC) 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 seen in fig. S2C. Georg E. Winter et al. Science 2015;348:1376-1381 Published by AAAS

Fig. 2 Chemical and genetic rescue of dBET1- and dFKBP-1–mediated degradation. Chemical and genetic rescue of dBET1- and dFKBP-1–mediated degradation. (A) Immunoblot for BRD4 and vinculin after a 4-hour pretreatment with 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−/− cells 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 pretreatment with 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 (Tub) after treatment of 293FTWT or 293FTCRBN−/− cells with dFKBP12 at the indicated concentrations for 18 hours. Georg E. Winter et al. Science 2015;348:1376-1381 Published by AAAS

Fig. 3 Selective BET bromodomain degradation established by expression proteomics. 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) 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) Selected proteins from (A) and (B) normalized to vehicle. Values represent triplicate means ± SD. (D) Immunoblot of BRD2, BRD3, BRD4, MYC, PIM1, and vinculin (VINC) after a 2-hour treatment of MV4;11 cells with DMSO, 250 nM dBET1, or 250 nM JQ1. (E) Quantitative reverse transcription–polymerase chain reaction 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. *P = 0.01 to 0.05; **P = 0.001 to 0.01; ****P < 0.0001. Georg E. Winter et al. Science 2015;348:1376-1381 Published by AAAS

Fig. 4 The kinetic and antileukemic advantage of BET bromodomain degradation. The kinetic and antileukemic advantage of BET bromodomain degradation. (A) 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) 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 phosphate-buffered saline (3 times) before cells were plated in drug-free medium 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 adenosine triphosphate–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) Tumor volume (means ± SEM) 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 (VINC) by using tumor lysates from mice treated either once for 4 hours or twice for 22 hours and 4 hours, compared with 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 three independent areas in fig. S8). (K) Percentage of mCherry+ leukemic cells (means ± SEM) 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. ***P = 0.0001 to 0.001; other P values as in Fig. 3 legend. Georg E. Winter et al. Science 2015;348:1376-1381 Published by AAAS