1. Volume 20, Issue 12, Pages 2860-2875 (September 2017)
Chemically Induced Degradation of the Oncogenic Transcription Factor BCL6 
Nina Kerres, Steffen Steurer, Stefanie Schlager, Gerd Bader, Helmut Berger, Maureen Caligiuri, Christian Dank, John R. Engen, Peter Ettmayer, Bernhard Fischerauer, Gerlinde Flotzinger, Daniel Gerlach, Thomas Gerstberger, Teresa Gmaschitz, Peter Greb, Bingsong Han, Elizabeth Heyes, Roxana E. Iacob, Dirk Kessler, Heike Kölle, Lyne Lamarre, David R. Lancia, Simon Lucas, Moriz Mayer, Katharina Mayr, Nikolai Mischerikow, Katja Mück, Christoph Peinsipp, Oliver Petermann, Ulrich Reiser, Dorothea Rudolph, Klaus Rumpel, Carina Salomon, Dirk Scharn, Renate Schnitzer, Andreas Schrenk, Norbert Schweifer, Diane Thompson, Elisabeth Traxler, Roland Varecka, Tilman Voss, Alexander Weiss-Puxbaum, Sandra Winkler, Xiaozhang Zheng, Andreas Zoephel, Norbert Kraut, Darryl McConnell, Mark Pearson, Manfred Koegl 
Cell Reports 
Volume 20, Issue 12, Pages (September 2017)
DOI: /j.celrep
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2. Cell Reports 2017 20, 2860-2875DOI: (10.1016/j.celrep.2017.08.081)
Copyright © 2017 Boehringer Ingelheim RCV GmbH & Co KG Terms and Conditions

3. Figure 1 Development of Potent Inhibitors of the BTB Domain of BCL6
(A) Compound optimization from screening hit 1 to BI-3802/3812.
(B–D) Co-crystal structures of small molecules binding to the co-repressor binding site that is formed at the interface of two BCL6 BTB domain monomers (B) 1, (C) 2, and (D) BI Green and blue represent the separate monomers of the BTB domain dimer. See Tables S1–S4 for crystallographic details.
(E) Chemical structure of the low-affinity BCL6 binder BI-5273 used as a negative control. Note that acidic compounds that are closely related to BI-5273 but are more potent on BCL6 readily entered cells and caused BCL6 inhibition.
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4. Figure 2 Effects of Atypical Compounds on the BTB Domain of BCL6
(A and B) The BCL6 inhibitor BI-3812 and an atypical inhibitor (BI-3802) were tested in cellular LUMIER assays for inhibition of the BCL6 co-repressor interaction (A) or the dimerization of the BTB domain of BCL6 (B), in both cases using an expression construct encompassing amino acids 1–373 of BCL6, including the BTB domain. Data are representative of two experiments done in triplicate. Error bars, SD.
(C) HDX MS data: Chiclet plot indicating the maximal difference in deuteration in BCL6 bound to the respective ligand versus free BCL6. Regions of interest upon binding are color coded corresponding to the representation in Figure 2D. Note that the difference seen in the peptide 31–47 and 33–47 were only seen with one of the two non-degrading compounds.
(D) HDX MS data plotted onto representations of the X-ray crystal structure of BI-3802 (green) color coded by the respective sequences as in Figure 2C. No significant effects upon compound binding are represented in gray areas. The approximate distance from BI-3802 to the red peptide is indicated. For every HDX experiment, two replicates were measured.
Deuterium incorporation graphs are depicted in Figure S1B.
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5. Figure 3 Compound-Induced Degradation of BCL6
(A) Cells (“C”) or cell lysates (“L”) from the indicated cell lines were treated for 60 min with 1 μM BI-3802, or left untreated (-) before analysis by western blotting. Data are representative of two experiments. See also Figure S2.
(B) Farage cells were treated with BI-3802, the proteasome inhibitor MG-132, or both and analyzed by western blotting for BCL6 (left and middle panel) or for multiubiquityl chains using TUBEs (right panel). For the middle and right panels, BCL6 was concentrated by immunoprecipitation prior to western blotting. Data are representative of two experiments. See also Figure S3.
(C) Time course of compound-induced degradation for complete and partial degraders in SU-DHL-4 cells. Cells were treated with saturating concentrations (5 μM) of the compounds indicated and analyzed for BCL6 levels by capillary electrophoresis after the time indicated. Error bars, SEM after normalization to GAPDH from three independent experiments done in duplicate. See also Figure S4.
(D) Effect of cycloheximide on the partial degradation of BCL6 induced by 7. SU-DHL-4 cells were treated with the compounds indicated, and BCL6 levels were quantified by capillary electrophoresis after the times indicated. Data are representative of two independent experiments done in triplicate. Error bars, SEM.
(E) Dose-dependent effects of a degrader (BI-3802) and a non-degrader (BI-3812) on BCL6 levels measured by capillary electrophoresis in DLBCL cell lines after 90 min of treatment in four different DLBCL cell lines. Data are representative of at least two independent experiments.
(F) Chemoaffinity pulldown of BCL6 with immobilized BI-3802 from lysate of Farage cells. Identified proteins are represented by dots, scaled by the number of unique peptides. Axes display their label-free intensity when compared to incubations with blank matrix from two independent experiments.
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6. Figure 4
Correlation of IC50 Values for BCL6 Degradation and Cellular Effects
(A) BCL6 degradation in SU-DHL-4 cells correlates with intracellular target engagement as measured by LUMIER assays for degraders. Colors represent degraders (green), partial degraders (yellow), and non-degraders (blue). “b” represents the slope; “r2” represents the coefficient of determination of the regression curve.
(B) Degradation efficiency of BCL6 in SU-DHL-4 cells at saturating concentrations of compounds measured as the percentage of protein degraded after 90 min (y axis) plotted against degradation DC50 (x axis). Note that more potent compounds (i.e., having low DC50 values) do not cause a higher fraction of BCL6 to be degraded than less potent compounds. No dose-response data were collected for compounds showing <50% degradation. Colors represent degraders (green), partial degraders (yellow), and non-degraders (blue).
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7. Figure 5 Domain Requirements for Compound-Induced Degradation of BCL6
Constructs depicted in the left panel were transiently transfected into HEK293 cells. P, PEST domain. The levels of BCL6 after 90 min of treatment with 500 nM BI-3802 were determined by western blotting. Expression of the constructs in the absence of BI-3802 was set as 100% for each constructs. Data are representative of at least two independent experiments. See also Figure S5.
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8. Figure 6 Effects of BCL6 Degradation on Gene Expression
(A) Volcano plot, x axis, log2 fold change induced by compound treatment compared to DMSO treatment, y axis, negative log10 p value for statistical significance. SU-DHL-4 cells were treated with 500 nM BI-3802 for 20 hr and 168 hr. Significantly induced genes (adj. p value ≤ 0.01, fold change ≥ 3) are depicted in blue and red for repressed and induced genes, respectively. Known BCL6-regulated genes are marked in green. Experiments were done in triplicates. See also Figure S6.
(B) Intersection of BCL6-bound genes in GC B cells (Huang et al., 2013; data from Cistrome DB [Mei et al., 2017] using a BETA score cutoff of 1 for putative target genes) with genes induced by BCL6 degradation (adj. p value ≤ 0.05, fold-change ≥ 2). A common set of 19 genes are found to intersect in SU-DHL-4 and Farage cells while being bound by BCL6 as shown by ChIP-seq in GC B cells. ∗∗∗p < 0.001; ∼not significant.
(C) Induction of BCL6-regulated genes by BI-3802 at early time points measured by qPCR in SU-DHL-4 cells. Data are representative of at least two independent experiments done in triplicate. Error bars, SD.
(D) Correlation of changes in gene expression in SU-DHL-4 cells treated with the degrader BI-3802 (x axes) and the non-degrader BI-3812 (y axes) compared to DMSO-treated cells after 20 hr and 168 hr of treatment. Regression lines as well as a line representing y = x are shown.
(E) Dose-dependent induction of BCL6 response genes was measured in SU-DHL-4 cells by qPCR in triplicate for a degrader (BI-3802), two partial degraders (6 and 7), and a non-degrader (BI-3812). Data are representative of two experiments.
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9. Figure 7
Antiproliferative Effects of the Degrader BI-3802 and the Non-degrader BI-3812 in DLBCL Cell Lines
(A) Long-term proliferation assays. Cells were kept at constant concentrations of the inhibitors as indicated and split to split to 200,000 cells per mL every 3–4 days. Split rates were multiplied to derive growth curves. Data are representative of at least two independent experiments. See also Figure S7 for additional cell lines.
(B) Inhibition of proliferation in Farage cells (y axis, IC50) correlates with BCL6 degradation in SU-DHL-4 cells (x axis, DC50) for degraders. Experiments to derive IC50 and DC50 values were done in triplicate.
(C) Inhibition of proliferation in Farage cells (y axis, IC50) correlates with intracellular target engagement for degraders as measured by the interaction of BCL6 and NCOR1 (x axis, IC50, LUMIER assays).
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