Article

Chemically Induced Degradation of the Oncogenic

Transcription Factor BCL6
Graphical Abstract Authors
Nina Kerres, Steffen Steurer,
Stefanie Schlager, ..., Darryl McConnell,
Mark Pearson, Manfred Koegl

Correspondence
manfred.koegl@boehringer-ingelheim.
com

In Brief
Kerres et al. show that the BTB domain of
BCL6 is highly druggable and that potent
binders can be derived that cause rapid
degradation of BCL6. Inhibitors that
induce degradation cause stronger anti-
proliferative effects than other BCL6
inhibitors and offer new routes to the
development of lymphoma treatments.

Highlights Accession Numbers

d The BCL6 BTB domain is a highly druggable structure with GSE94099
inhibitor IC50 values <3 nM

d Some compounds cause rapid degradation of BCL6 via the

ubiquitin system

d Loss of BCL6 causes de-repression of target genes and

curbs proliferation

d BCL6 degradation has stronger effects than BCL6 inhibition

Kerres et al., 2017, Cell Reports 20, 2860–2875

September 19, 2017 ª 2017 Boehringer Ingelheim RCV GmbH & Co KG.
http://dx.doi.org/10.1016/j.celrep.2017.08.081
 Cell Reports

Article

Chemically Induced Degradation of the Oncogenic

Transcription Factor BCL6
Nina Kerres,1 Steffen Steurer,1 Stefanie Schlager,1 Gerd Bader,1 Helmut Berger,1 Maureen Caligiuri,2 Christian Dank,1
John R. Engen,3 Peter Ettmayer,1 Bernhard Fischerauer,1 Gerlinde Flotzinger,1 Daniel Gerlach,1 Thomas Gerstberger,1
Teresa Gmaschitz,1 Peter Greb,1 Bingsong Han,2 Elizabeth Heyes,1 Roxana E. Iacob,3 Dirk Kessler,1 Heike Kölle,4
Lyne Lamarre,1 David R. Lancia,2 Simon Lucas,1 Moriz Mayer,1 Katharina Mayr,1 Nikolai Mischerikow,1 Katja Mu € ck,4
Christoph Peinsipp,1 Oliver Petermann,1 Ulrich Reiser,1 Dorothea Rudolph,1 Klaus Rumpel,1 Carina Salomon,1
Dirk Scharn,1 Renate Schnitzer,1 Andreas Schrenk,1 Norbert Schweifer,1 Diane Thompson,1 Elisabeth Traxler,1
Roland Varecka,1 Tilman Voss,1 Alexander Weiss-Puxbaum,1 Sandra Winkler,1 Xiaozhang Zheng,2 Andreas Zoephel,1
Norbert Kraut,1 Darryl McConnell,1 Mark Pearson,1 and Manfred Koegl1,5,*
1Boehringer Ingelheim RCV GmbH & Co KG, 1221 Vienna, Austria
2FORMA Therapeutics, Watertown, MA 02472, USA
3Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, USA
4Boehringer Ingelheim, MedChem, Structural Research, Birkendorfer Str. 65, 88397 Biberach, Germany
5Lead Contact

*Correspondence: manfred.koegl@boehringer-ingelheim.com
http://dx.doi.org/10.1016/j.celrep.2017.08.081

SUMMARY The transcription factor BCL6 is required for the GC reaction

The transcription factor BCL6 is a known driver of
oncogenesis in lymphoid malignancies, including
diffuse large B cell lymphoma (DLBCL). Disruption
of its interaction with transcriptional repressors inter-
feres with the oncogenic effects of BCL6. We used
a structure-based drug design to develop highly
potent compounds that block this interaction. A sub-
set of these inhibitors also causes rapid ubiquityla-
tion and degradation of BCL6 in cells. These com-
pounds display significantly stronger induction of
expression of BCL6-repressed genes and anti-prolif-
erative effects than compounds that merely inhibit
co-repressor interactions. This work establishes the
BTB domain as a highly druggable structure, paving
the way for the use of other members of this protein
family as drug targets. The magnitude of effects
elicited by this class of BCL6-degrading compounds
exceeds that of our equipotent non-degrading inhib-
itors, suggesting opportunities for the development
of BCL6-based lymphoma therapeutics.
INTRODUCTION
Germinal centers (GCs) are substructures of lymph nodes that are
dedicated to the selection of B cells expressing high-affinity anti-
bodies (Basso and Dalla-Favera, 2015; Mesin et al., 2016). In the
GC reaction, B cells undergo cycles of fast cell division, somatic
hypermutation of their immunoglobulin genes, and selection for
high-affinity antibody expression via interaction with follicular
T helper cells. B cells producing high-affinity antibodies can leave
the GCs and mature to antibody-producing plasma cells or mem-
ory B cells. Accidental mutations in the GC reaction can give rise
to mutated B cells that maintain an elevated proliferation and fail
to differentiate, contributing to the genesis of DLBCL.
(Dent et al., 1997; Ye et al., 1997). BCL6 represses the expres-
sion of a broad set of genes that are required to sustain muta-
genic activity without activating the DNA damage response or
apoptosis (Basso et al., 2010; Ci et al., 2009). BCL6 also prevents
maturation to plasma or memory cells and helps to maintain a
de-differentiated state. Its expression must be switched off to
allow the B cell to exit the GC cycle and differentiate.
BCL6 is an oncogenic driver for DLBCL (Basso and Dalla-Fa-
vera, 2012; Hatzi and Melnick, 2014; Pasqualucci, 2013). Its
expression is frequently elevated by mutations in DLBCL.
Recurrent mutations leading to elevated BCL6 include translo-
cations (Ye et al., 1993), promoter mutations (Migliazza et al.,
1995; Pasqualucci et al., 2003), as well as mutations in regula-
tors of BCL6 expression or stability (Duan et al., 2012; Pasqua-
lucci et al., 2011; Ying et al., 2013). Dysregulated expression of
BCL6 in mice leads to lymphomagenesis (Cattoretti et al.,
2005).
BCL6 functions as a transcriptional repressor that binds
specific DNA sequences via its zinc fingers (ZFs) and recruits
transcriptional co-repressor complexes by its BTB/POZ
(Broad-Complex, Tramtrack, and Bric a brac [Zollman et al.,
1994] or poxvirus and ZF [Bardwell and Treisman, 1994])
domain. Mutation of the co-repressor binding interface on
BCL6 has been shown to prevent the formation of GCs (Huang
et al., 2013) but lacks the pathological inflammatory defects
associated with germline deletion of BCL6 (Dent et al., 1997;
Ye et al., 1997). Thus, pharmacological inhibition of the interac-
tion of BCL6 with co-repressor proteins promises to block the
oncogenic function of the protein with few or no side effects
other than the lack of affinity maturation of B cells.
The BTB domain is a protein interaction motif found at the
N terminus of several ZF transcription factors, potassium chan-
nels, and E3 ligase subunits. It forms a tightly intertwined
head-to-tail homodimer (Ahmad et al., 1998) and can adopt a
variety of oligomeric states, e.g., a pentameric state as seen
for potassium channels (Ji et al., 2016). Each dimer has two iden-
tical interfaces at the junction of the two subunits, which, in the

2860 Cell Reports 20, 2860–2875, September 19, 2017 ª 2017 Boehringer Ingelheim RCV GmbH & Co KG.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
case of BCL6, can bind to sequences found on transcriptional
co-repressors, such as NCOR1, SMRT, and BCOR. Co-
repressor peptides bind in an extended conformation to a groove
formed at the dimer interface (Ahmad et al., 2003; Ghetu et al.,
2008). Low-affinity non-peptidic inhibitors of the BTB domain
of BCL6 have been reported (Cerchietti et al., 2010), the most
potent compounds displaying half-maximal cellular activities at
around 35 mM (Cardenas et al., 2016).
Protein levels of BCL6 are subject to control by regulated
degradation. Activation of B cell receptor signaling causes
mitogen-activated protein (MAP) kinase-dependent phosphory-
lation and degradation of BCL6 (Niu et al., 1998). Genotoxic
stress has been shown to cause phosphorylation of BCL6 by
ATM and ATR kinases and ubiquitin-dependent degradation of
BCL6 (Phan et al., 2007). In GC cells, BCL6 degradation requires
the E3 ligase FBXO11. Mutational inactivation of FBXO11 has
been shown to stabilize BCL6 and is recurrently observed in
DLBCLs (Duan et al., 2012). Mice lacking FBXO11 in the B cell
lineage display expanded dark zones in GCs, higher levels of
BCL6 protein, and development of lymphoproliferative disorders
(Schneider et al., 2016).
Here, we show that the BTB domain of BCL6 is highly
druggable and allows the development of potent inhibitors.
We used a structure-based drug design to generate BCL6-
binding compounds with nanomolar affinities. Surprisingly, a
subclass of these structures induces ubiquitylation and fast
proteasome-dependent degradation of BCL6. Chemically
induced degradation of BCL6 causes de-repression of
BCL6-bound genes and has antiproliferative effects in several
DLBCL cell lines. We use equipotent compounds of BCL6
degrading and non-degrading inhibitors to examine the
effects of BCL6 inhibition versus BCL6 protein loss with
regard to transcriptional and antiproliferative effects in lym-
phoma cell lines.
RESULTS
Screening
A high throughput screen was performed with a library of
around 1,700,000 compounds using a fluorescence polarization
(FP) assay monitoring the interaction of a co-repressor peptide
with the BTB domain of BCL6, resulting in a hit rate of 0.013%.
202 confirmed active compounds were clustered according to
similarity; cluster representatives were repurified and confirmed
in the FP assay. 31 confirmed hits were tested for dose-depen-
dent and saturable binding to BCL6 by surface plasmon reso-
nance (SPR). Nine of these 31 hits could be further confirmed,
and five of these could be co-crystalized with the BTB domain
of BCL6. 1 was selected as a promising starting point based on
its potential for further optimization (scaffold exit vectors
pointed directed toward unoccupied regions on the binding
pocket) and binding affinities (FP concentration of half-maximal
inhibition (IC50) = 18 mM; SPR dissociation constant [KD] =
20 mM) (Figure 1A; Table 1). While the initial hits were character-
ized mainly by the FP assay, further characterization of the
compounds was done by a biochemical ULight co-repressor
peptide-binding assay in addition to the SPR biophysical
method.
Structure-Activity Relationship of 4-Amino-5-Chloro-
Pyrimidines Binding to BCL6
The co-crystal structure of 1 (Figure 1B) reveals the ligand bind-
ing to the lipophilic cavity of the co-repressor binding site formed
at the interface of two BCL6 BTB domain monomers. The main
hydrophilic interaction of this hit is the anilinic H-bond donor
interacting with Met51. This polar anchor leads to the chloropyr-
imidine being buried deeply in the lipophilic subpocket and
places the pyrazole in an approximate distance of 3.6 Å to
Arg24, the pyrazole acting as an H-bond acceptor for the flexible
side chain of Arg28. The chlorophenyl residue a rests on a rela-
tively flat and open part of the pocket and offers a vector for
further optimization of binding affinity.
Optimization of Pyrimidine-R4
Binding affinity of the initial hit 1 according to FP and ULight
assays, respectively, could be improved by an order of magni-
tude by exchanging the chlorophenyl a for an indolinone b,
which anchors the ligand to the Glu115 backbone NH with its
lactam carbonyl (Figures 1B and 1C; Table 1). 6-membered
analogs of this H-bond acceptor motif such as dihydroquinoline
derivative c displayed comparable ligand efficiencies, but the
6-membered ring analogs such as c were prioritized over the in-
dolinones because of a favorable vector toward the polar
groove of the binding site. A further factor of 40 improvement
of binding affinity (e.g., comparing 3 and 4, Table 1) was
achieved with the planar oxyquinoline motif e. The potency
improvement can be explained by the acetamide side chain
of e adding a polar interaction to the backbone NH of Val117
at a heavy atom distance of 3.6 Å as well as positioning the
a-carbon of the side chain at a 3.3 Å distance to the backbone
carbonyl of Cys53 (Figure 1D). The methoxy derivatives d, de-
signed to increase the interaction to Tyr58, were ambiguous
in terms of potency, gaining or losing binding affinity in different
R2 subseries compared to e.
Optimization of Pyrimidine-R2
Exploring the structure-activity relationship (SAR) in the region of
the two arginine side chains (R24 and R28), binding affinity could
be only modestly improved through modification of the dimethyl-
pyrazole to differently substituted aromatic rings, piperidines,
piperazines, and various fused ring systems. In co-crystal struc-
tures of different R4s with the same dimethylpyrazole ligand, the
orientation of the dimethylpyrazole ring to both Arg24 and Arg28
showed a lot of variety (e.g., compare Figures 1B and 1C).
Consistently, both Arg24 and Arg28 showed a lot of flexibility
comparing different co-crystal structures. Saturated R4 motifs
were prioritized over aromatic residues due to superior affinity
and physicochemical properties. From dimethylpyrazole f via
dimethylpiperidine N-carboxamide g to difluoropiperidine i or
dimethylpiperidine h, the binding affinity improved by approxi-
mately a factor of 2 in each case (Figures 1A and 1B; Table 1,
compare 4, 5, 6, BI-3802).
A Subset of Compounds Induces Ubiquitylation and
Proteasome-Dependent Degradation of BCL6
We noticed that a small subset of compounds behaved atypi-
cally in biophysical assays that use very high protein concentra-
tions (>100 mM). They consistently led to protein precipitation in
co-crystallization and protein nuclear magnetic resonance
Cell Reports 20, 2860–2875, September 19, 2017 2861
 A

B C

D E

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-3802. 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.

2862 Cell Reports 20, 2860–2875, September 19, 2017

Table 1. Binding Affinity of Compounds 1–7, BI-3812, BI-3802, Not surprisingly, areas close to the binding site (amino acids
and BI-5273 in Biochemical (FP, ULight) and Cellular-Binding 48–54, 108–117, and 101–107, color coded in orange, blue,
Assays (LUMIER) and turquoise, Figures 2C and 2D), which show a high degree
FP ULight LUMIER of exchange in the absence of compound, were protected to a
Compound No. R2 R4 IC50 (nM) IC50 (nM) IC50 (nM) great extent from HDX upon binding of all compounds tested
1 f a 18,648 >20,000a – (Figures 2C and S1B). The atypical compounds showed slightly
more protection than the typical compounds in a region close to
2 f b 1,918 632 9,614
the binding site (amino acids 18–30, yellow). Interestingly, the
3 f c 2,285 471 4,443
atypical compounds also induced significantly more protection
4 f e – 12a 58 in an additional area farther remote from the binding site (amino
5 g e – 5a 154 acids 69–80, red). This difference between the two groups high-
6 i e – 4a 128 lights a difference in stabilization effects between the two
7 j e – 4a 57 subsets of compounds. It is conceivable that protection from
BI-3802 h e – %3 a
43 HDX in areas outside of the ligand-binding domain is brought
BI-3812 g d – %3a 40 about by BCL6 dimer-dimer interactions being facilitated in the
atypical group. Indeed, dimer-dimer interactions and inducible
BI-5273 h – 10,162a –
a aggregation have been described for other BTB domains (Li
ULight LOW (for a more detailed property description of probe com-
et al., 1999; Liu et al., 2016; Stead et al., 2007). We therefore
pounds BI-3812 and BI-3802, see Table S4). Compounds BI-3812 and
BI-3802 inhibit the BTB domain of BCL6 in the low nanomolar range.
wondered whether these differences in behavior on a molecular
With affinities of approximately 3 nM, the assay wall of the ULight assay level would translate into cellular effects in lymphoma cells.
is reached, limiting the accuracy of the biochemical assay for these most When we examined the effects of BCL6 inhibitors on cells, we
potent BCL6 inhibitors. noted that the atypical subset of compounds caused BCL6
protein to become undetectable after treatment. This effect
was observed in ten out of ten lymphoma cell lines tested (Fig-
(NMR) experiments. However, these compounds could be ures 3A, 3E, and S2). The addition of the compound to the lysate
successfully soaked into BCL6 crystals and behave inconspicu- had no effect on BCL6 levels, indicating that the effect depends
ously in cellular experiments testing for binding of BCL6 to co-re- on cellular integrity (Figure 3A).
pressors (LUMIER, luminescence-based mammalian intractome The disappearance of BCL6 could be attributed to protea-
[Barrios-Rodiles et al., 2005] assays; see Experimental Proced- some-dependent protein degradation, since it was reversible
ures). To exclude that these compounds simply denature by inhibition of the proteasome with MG-132 (Figures 3B and
BCL6, we examined their effects on dimerization of BCL6 as S3). After concomitant treatment with a BCL6-binding com-
measured in LUMIER experiments. While these compounds pound and MG-132, the appearance of more slowly migrating
effectively inhibited the binding of the BTB domain of BCL6 to forms of BCL6 was notable, suggestive of multi-ubiquitylation.
co-repressors, they did not inhibit the dimerization of the BTB To test for the presence of multi-ubiquitin chains, we probed
domain (Figures 2A and 2B). While not formally showing that an immune precipitate of BCL6 with tandem ubiquitin-binding
the dimerization was in the native form, this argued against entities (TUBEs). Treatment with the degradation-inducing com-
protein denaturation as an explanation for their atypical pound BI-3802 caused the appearance of multi-ubiquitin chains
behavior. Additionally, in differential scanning fluorimetry mea- on BCL6, which was strongly augmented by inhibition of the
surements, upon the addition of both BI-3802 and BI-3812, a proteasome. We conclude that some BCL6-binding compounds
melting point of the BTB domain of BCL6 was still apparent cause disappearance of BCL6 from cells via the induction of
and its thermal stability was increased (Figures S2C and S2D), ubiquitylation and proteasome-dependent degradation.
which is not compatible with the assumption of major unfolding Degradation was remarkably fast, reducing BCL6 levels by
events induced by the compounds. half in about 5 min (Figures 3C and S4), and by R90% in 2 hr.
We compared four compounds, including two atypical ones The effect was dose dependent, and concentrations of half-
(Figure S1A), in a hydrogen deuterium exchange mass spec- maximal degradation (DC50 values) were used to describe
trometry (HDX MS) experiment to probe for regions that are degradation potency. DC50 values derived from these cellular
affected by compound binding (Figures 2C, 2D, and S1B). HDX assays and IC50 values derived for inhibition of BCL6 binding
MS probes protein structure and interactions by measuring the to co-repressor peptides in cellular assays show a tight correla-
deuterium incorporation of backbone amide hydrogens (Engen, tion (Figure 4A). This strongly indicates that degradation is
2009). When diluted into deuterium buffer, backbone hydrogens brought about by direct binding of the compounds to BCL6,
of flexible and/or exposed protein regions rapidly exchange with and not by indirect effects. When examined across a spectrum
deuterium, whereas buried domains and/or those regions that of compounds, it became apparent that the maximally induced
contain hydrogen bonding involving backbone amide hydrogens degradation (degradation efficiency) varies widely. For clarity,
demonstrate slowed or suppressed deuterium exchange. we will refer to the compounds reducing BCL6 levels by greater
Changes in deuterium levels, for example, upon binding of a than 70% as ‘‘degraders,’’ and to the other compounds as ‘‘non-
ligand, indicate protection/exposure of backbone amide hydro- degraders’’ (<30% reduction) or ‘‘partial degraders’’ (30%–70%
gens to the deuterium solvent or alterations to the hydrogen- reduction). Partial degraders lead to a limited reduction of BCL6
bonding network. levels even after prolonged incubation at saturating doses and

Cell Reports 20, 2860–2875, September 19, 2017 2863

 A C
BI-3802 BI-3812
Colour
BCL6-NCOR1
code
Normalised Signal/Background

1.5

1.0
BI-3802
BI-3812
0.5

0.0
-4 -3 -2 -1 0 1 2
log µM cpd

BCL6-BCL6
Normalised Signal/Background

1.5

BI-3802
1.0
BI-3812

0.5

0.0
-4 -3 -2 -1 0 1 2
log µM cpd

90°

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.

(legend continued on next page)

2864 Cell Reports 20, 2860–2875, September 19, 2017

degradation of BCL6 proceeded more slowly (Figure 3C).
Concomitant inhibition of protein synthesis caused a partial
degrader to induce complete degradation of BCL6 (Figure 3D).
Conceivably, the remaining fraction of BCL6 is determined by
the equilibrium of degradation and re-synthesis, with com-
pounds inducing a faster degradation rate reaching an equilib-
rium at lower levels of BCL6.
Degradation efficiency did not correlate with degradation
potency (i.e., DC50 values, Figure 4B). Thus, there is a qualitative
difference between compounds that do or do not induce BCL6
degradation that is not linked to affinity. Linking the degradation
effect to the architecture of the inhibitors revealed that the pyrim-
idine-R2 residue plays an especially crucial role. In BI-3802, this
residue is solvent exposed and makes no direct interaction with
the protein (Figures 1D and 2D). As shown in Table 2, variation of
that position can make the difference for a compound being a
degrader, a partial degrader, or a non-degrader. Polar or
charged moieties (compounds 5, 9, 10) generally result in non-
degraders, whereas the degraders are characterized by having
non-polar and more lipophilic uncharged residues at this posi-
tion (BI-3802 and compounds 6, 8). Lipophilicity alone is insuffi-
cient, however, since not every lipophilic substituent caused
degradation (e.g., compound 11, Table 2). The degrader
BI-3802 was selected for further examination of the cellular
effects, as well as one equipotent structurally closely related
non-degrader, BI-3812 (Table 1; Figure 1A). The selectivity
profile of BI-3802 was determined in a chemoaffinity pulldown
with an immobilized BI-3802 analog (Figure 3F). BCL6 was
confirmed as the major target of this compound in DLBCL (Far-
age) cells; of note, no other BTB/POZ domain-containing
proteins were identified.
Compound-Induced Degradation of BCL6 Requires a
Functional DNA-Binding Domain
We noted that the truncated constructs of BCL6 used in LUMIER
assays that lack the ZF DNA-binding domain do not disappear
after treatment with degraders. To study the domains required
for degradation of BCL6 in further detail, we employed
HEK293 cells that lack endogenous BCL6. Degradation of
BCL6 induced by activation of B cell receptor signaling depends
on MAP kinase-dependent phosphorylation of serines 333 and
343 and the presence of a sequence that is rich in proline, gluta-
mic acid, serine, and threonine (PEST) elements between amino
acids 300 and 417 (Niu et al., 1998). A construct that had 300
amino acids of the region separating the BTB domain from the
ZF deleted and therefore lacked both the PEST sequences as
well as the MAP kinase phosphorylation sites was efficiently
degraded (Figure 5). In agreement with this finding, inhibition of
MEK by trametinib had no influence on compound-induced
degradation of BCL6 (Figure S5). Genotoxic stress has been
(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.
shown to destabilize BCL6 protein dependent on phosphoryla-
tion of BCL6 by ATM/ATR kinases (Phan et al., 2007). Com-
pound-induced BCL6 degradation was not influenced by
inhibition of ATM, ATR, or both kinases (Figure S5). Thus,
compound-induced degradation occurs via a different pathway
than the ones induced by activated B cell receptor signaling or
genotoxic stress. Degradation also occurred independent of
the known FBXO11-dependent degradation pathway, as it was
also observed in Farage cells that lack FBXO11, as well as
OCI-Ly1 cells in which FBXO11 is inactivated by mutation (Fig-
ure 3E) (Duan et al., 2012).
In contrast, deletion or mutation of the ZF resulted in complete
loss of the degradation effect. This suggests that either structural
elements within the ZF or its capacity to bind DNA are required to
support degradation. To distinguish between these possibilities,
we attached the BTB domain of BCL6 to DNA-binding domains
from different transcription factors. The ZF domain of ESRRA
partially restored compound-induced BTB domain-dependent
degradation, and the basic helix-loop-helix domain of TCF4
completely restored degradation (Figure 5). The fact that an iso-
lated BTB domain that lacks a DNA-binding domain does not get
destabilized argues against compound-induced unfolding of the
BTB domain as the main reason for degradation, but it points to a
more selective degradation mechanism that requires localization
on DNA. Thus, we conclude that the structure of the BCL6 ZF is
dispensable for degradation, but binding to DNA is required.
BCL6-Degrading Compounds Induce Expression of
BCL6-Repressed Genes More Potently Than Non-
degrading Inhibitors
We compared the effects of a degrader and a non-degrader on
the de-repression of BCL6 target genes in DLCBL cells. In
SU-DHL-4 cells, the degrader BI-3802 induced the expression
of 87 genes after 20 hr of treatment, whereas only 17 genes
were reduced in expression, in line with the role of BCL6 as a
transcriptional repressor (Table S6A). Several of the known
BCL6-regulated genes were significantly induced, including
ATM, PRDM1, PTPN6, CD69, IRF4, and DUSP5 (Figure 6A;
Tables S6B and S6C). Induced genes after 7 days significantly
overlapped with genes that have proximal BCL6-binding sites
as identified by ChIP in GC B cells (Figure 6B; Tables S6D and
S6E) (Huang et al., 2013), with 95% being within 5 kb of the tran-
scriptional start site. Three genes were selected based on their
robust induction and proximity to BCL6-binding sites. For these
genes, de-repression was detectable already 4 hr after com-
pound treatment, compatible with a direct effect (Figure 6C).
Genes upregulated by the degrader BI-3802 were generally
also upregulated by the non-degrader BI-3812, but to a lesser
extent, as can be seen in Figure 6D. While there was a significant
correlation of the effects of BI-3802 with the effects of BI-3812,
Cell Reports 20, 2860–2875, September 19, 2017 2865
 A B

C 1 .5 D

1.0

Fraction remaining
B I- 3 8 1 2
F r a c t io n r e m a in in g

1 .0

0.5 7
0 .5 Cycloheximide
7
7 + Cycloheximide
6

B I- 3 8 0 2
0.0
0 .0 0 2 4 6 8
0 200 400

M in u te s Hours

E Farage OCI-Ly1 BI-3802 F

Normalised BCL6 protein level

Normalised BCL6 protein level

1.5 1.5 BI-3812

1.0 1.0

BCL6
Inhibitor beads / control beads (assay 2)

0.5 0.5
1

0.0 0.0
-4 -3 -2 -1 0 1 2 -4 -3 -2 -1 0 1 2

log µM cpd log µM cpd

SU-DHL-4 BJAB
10
Normalised BCL6 protein level

Normalised BCL6 protein level

1.5 1.5

1.0 1.0

0.5 0.5

0.0 0.0
-4 -3 -2 -1 0 1 2 -4 -3 -2 -1 0 1 2
100
log µM cpd log µM cpd

1 10 100
Inhibitor beads / control beads (assay 1)

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 mM 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 mM) 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.

2866 Cell Reports 20, 2860–2875, September 19, 2017


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 concen-
trations 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).
the amplitude of the effect was consistently larger for the
degrader, as visualized by the regression line lying below the di-
agonal (y = x) line. This was also apparent when selected genes
were tested by qPCR for dose-dependent induction. While the
two compounds exerted their effects at comparable IC50 values,
the extent of induction was significantly greater for the degrader
Table 2. Structure-Activity Relationship for the Induced
Degradation of BCL6
Percent
Degradation
ULight Low after 90 min
Compound No. R2 R4 IC50 (nM) (c = 5 mM) Classification
BI-3802 h e %3 80 degrader
6 i e 4 56 partial degrader
8 k e 6 52 partial degrader
5 g e 5 – non-degrader
9 m e 9 22 non-degrader
10 l e 4 – non-degrader
11 n e 10 – non-degrader
(Figure 6E). Compounds causing only a partial reduction of BCL6
protein levels, such as 6, induced these genes to higher levels
than non-degraders but to lower levels than compounds that
cause complete degradation of BCL6 (Figure 6E). This difference
in the effect of degrading versus non-degrading compounds was
consistent across a large set of compounds. The observation is
in agreement with additional repressive effects that are brought
about by sequences of BCL6 outside of the BTB domain (Huang
et al., 2014), which are affected by a degrader but unaffected by
a non-degrader. Whereas the same correlation of the effects of
BI-3802 and BI-3812 was apparent in Farage cells after 20 hr
of treatment, only a small number of genes were affected. After
7 days of treatment, the overlap of genes induced in Farage cells
was highly significant with those induced in SU-DHL-4 cells (78
of 172 genes overlap, p < 0.001, Figures 6B and S6A). The
gene most significantly de-repressed by BI-3802 in Farage cells
after 7 days of treatment was PRDM1/Blimp-1, a known BCL6-
repressed gene and master regulator of plasma cell differentia-
tion. The transcriptional effects of the degrader BI-3802, the
non-degrader BI-3812, as well as the negative control com-
pound BI-5273 on BCL6 target genes were measured in a panel
of 5 DLBCL cell lines, plus one that expresses only minimal
amounts of BCL6—Toledo (Figure S6B). BI-5273 is related in
structure but does not potently bind to BCL6, displaying an
IC50 of 10 mM in the ULight assay. For seven of the eight genes,
a significant induction in two or more of the cell lines was
observed with BI-3802 and BI-3812, but not BI-5273, the effects
on Toledo always being the lowest or missing. In all cases, the
effect of the degrader was the strongest; the non-degrader
had weaker or no effects; and the negative control compound
was inactive.
Degradation of BCL6 Results in Growth Arrest with
Slow-Onset Kinetics
Treatment with BCL6 degraders had antiproliferative effects of
variable degrees in several DLBCL cell lines (Figures 7A and
S7). The responses had a slow onset, with the cells continuing
to cycle for 4–7 days before stasis was reached, reminiscent of
the delayed growth inhibitory activities of compounds inhibiting
chromatin regulators such as EZH2 (Knutson et al., 2012). In
some BCL6-positive lines, cells persisted but grew at a reduced
growth rate; in others, such as OCI-Ly19, there was no
Cell Reports 20, 2860–2875, September 19, 2017 2867
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 repre-
sentative of at least two independent experiments. See also Figure S5.
measurable response. We failed to detect significant signs of
apoptosis induction in cells depleted of BCL6. Non-degraders
such as BI-3812 were antiproliferative only at concentrations
far above their IC50 of BCL6 inhibition. Cells not expressing
BCL6, such as MV-4-11 cells and Toledo cells, were insensitive
to BI-3802. The structurally related low-affinity BCL6 binder BI-
5273 had no effect on DLBCL proliferation (Figure 7A). The anti-
proliferative potencies of a large set of compounds were tested
in Farage cells. IC50 values are tightly correlated with the DC50
values for BCL6 degradation (Figure 7B) and inhibition of
co-repressor binding (Figure 7C), strongly arguing for a BCL6-
mediated effect as opposed to off-target activities. In summary,
compound-induced degradation of BCL6 had clear antiprolifer-
ative consequences in several DLBCL cell lines, but it did not
significantly induce cell death.
While the poor bioavailability of BI-3802 limits its use in animal
studies (Table S5), these results show that BI-3802 is a highly
potent and efficacious BCL6 degrader probe compound and
BI-3812 is a highly potent and efficacious BCL6 inhibitor probe
compound. They will allow dissection of the effects of BCL6
inhibition and/or degradation on BCL6-positive lymphoprolifera-
tive disorders and can be used in ex vivo studies on the behavior
of BCL6-dependent cells, such as GC helper B and follicular
helper T cells.
DISCUSSION
The BTB Domain of BCL6 Is Highly Druggable
This work shows that the BTB domain of BCL6 is highly drug-
gable, and while weakly potent inhibitors of this domain have
been reported (Cardenas et al., 2016; Cerchietti et al., 2010),
our study shows that inhibitors can be generated that are at least
three orders of magnitude more potent and thus reach the
potency range required for biological investigation and ultimate
use as a therapeutic in humans. Given the structural conserva-
tion within the family, it is highly likely that druggability is a family
trait and many, if not most, BTB domains can be targeted by
2868 Cell Reports 20, 2860–2875, September 19, 2017
small molecules. Indeed, the redox-sensing protein KEAP1 is
known to respond to electrophiles, and structural studies have
shown covalvent (Cleasby et al., 2014) as well as non-covalent
(Huerta et al., 2016) binding of ligands to its BTB domain. While
this paper was in review, two papers were published that report
the identification of chemical ligands for the BTB domain of
BCL6 (Kamada et al., 2017; McCoull et al., 2017). It is not clear
if these compounds promote the degradation of BCL6. Since
many BTB-domain proteins have pivotal roles in transcriptional
regulation and ubiquitin-dependent degradation (Chaharbakhshi
and Jemc, 2016; Perez-Torrado et al., 2006; Siggs and Beutler,
2012), it appears likely that further drug targets will emerge
from this family. It remains to be seen if degradation-inducing
compounds can be found for BTB-domain proteins other than
BCL6.
Chemically Induced Protein Degradation
Induced degradation of transcription factors is not unprece-
dented: like BCL6, estrogen receptors can be inhibited by antag-
onists such as tamoxifen that modulate the interaction of the
receptor with transcriptional activators (Jordan, 2003). Com-
pounds that cause proteasome-dependent receptor degrada-
tion, such as fulvestrant (Osborne et al., 2004), shut down all
transcriptional activity of estrogen receptors and remain a treat-
ment option in breast carcinomas that have progressed after
tamoxifen therapy (Di Leo et al., 2010). Recently, chemically
induced degradation of proteins by small molecules that bridge
the target protein to the ubiquitin-dependent degradation
machinery has become an emerging field in drug discovery
(Toure and Crews, 2016). These PROTACs (proteolysis targeting
chimeric molecules) have been shown to surpass traditional
inhibitors, e.g., for the inhibition of BET family proteins (Lu
et al., 2015; Raina et al., 2016; Winter et al., 2015). We report
here the discovery of a small molecule that similarly directs
degradation of its target protein, BCL6. The observation that
the mere inhibition of the BCL6 BTB domain has milder effects
on transcription of repressed genes than the removal of the
 (legend on next page)

Cell Reports 20, 2860–2875, September 19, 2017 2869

whole protein is reminiscent of the stronger effects that
PROTACs have compared to the parent inhibitors. These obser-
vations are in line with reports showing that regions outside of the
BTB domain of BCL6 also partake in the transcriptional repres-
sion necessary for the physiological function of BCL6 (Huang
et al., 2014; Nance et al., 2015). While the effects of a degrader
are stronger than are those of a non-degrading inhibitor and
responding genes are enriched for genes that have a BCL6-bind-
ing site in their vicinity, they still only affect a very limited number
of genes. This narrow impact argues against non-specific effects
of the compounds on protein stability, which would be expected
to cause a more broad response related to other protein-dena-
turing stress responses not tied to BCL6, such as the heat-shock
response.
Some BTB domains are known to bind to cullins to direct
protein ubiquitylation and degradation (Furukawa et al., 2003;
Geyer et al., 2003; Xu et al., 2003). Since the induced degrada-
tion we observed was insensitive to MLN4924, a general inhibitor
of cullin-dependent protein ubiquitylation (Soucy et al., 2009),
this mechanism is unlikely for BCL6, and the pathway respon-
sible for compound-induced degradation of BCL6 remains to
be elucidated.
Antiproliferative Effects of BCL6 Degradation
The loss of BCL6 protein induced by compounds such as
BI-3802 had antiproliferative effects in several DLBCL cell lines.
These effects took several days to manifest and did not lead to
cell death, but they caused cells simply to continue proliferation
at a slower pace. The induction of genes associated with B cell
maturation such as PRDM1/Blimp-1 and IRF4, albeit to low
levels, suggests that this phenomenon may be related to the
induction of a partially differentiated state. The availability of a
potent tool compound for in vitro studies such as BI-3802 will
permit dissection of these responses in greater detail in future
studies. To probe whether such an effect can be exploited ther-
apeutically, it will be of interest to compare the responses
induced in cell lines with those of primary lymphoma cells.
In mouse models, a complete knockout of BCL6 results in a
severe inflammatory phenotype, whereas mutation of the core-
pressor binding site on BCL6 does not cause such a phenotype
and only blocks the formation of GCs. In this respect, it is note-
worthy that in our cell line studies, both BCL6 degraders and
inhibitors affect a similar repertoire of genes, with the degraders
simply displaying stronger effect sizes than the inhibitors. It is
important to stress that only BCL6-degrading compounds effec-
tively curbed proliferation. Compounds that inhibited the binding
of BCL6 to co-repressors with identical potencies, but did not
cause BCL6 degradation, only had marginal effects on prolifera-
tion. Thus, the advent of BCL6-degrading small molecules
potentially offers a novel path forward for the development of
new therapeutics for the treatment of BCL6-driven lymphomas.
EXPERIMENTAL PROCEDURES
Protein Purification, Crystallization, Hydrogen-Deuterium
Exchange, SPR KD Assay, and LUMIER Assays
For protein crystallization, SPR studies, HDX experiments, FP, and ULight
assays, a protein fragment encompassing the BTB domain of BCL6 purified
from Escherichia coli (E. coli) was used. LUMIER assays were done as
described (Barrios-Rodiles et al., 2005; Blasche and Koegl, 2013). See Supple-
mental Experimental Procedures for details.
BCL6-BCOR FP Assay
For the assay-ready plates used in this assay, the DMSO compound stock
solutions are directly transferred to 384-well nonbinding surface (NBS)-
coated plates (Corning 3820, black). For screening and single-dose confir-
mation assay-ready plates, a volume of 100 nL of a 2 mM DMSO stock
was transferred to the assay plates. For dose response and IC50 assay plates,
250 nL of an 8 point 2-fold serial dilution from a 2 mM stock in DMSO was
transferred. All reagent solutions were prepared in assay buffer (1 mM HEPES
[pH 7.4], 5 mM NaCl, 0.01% Triton X-100, 10 mM L-glutathione), and all ad-
ditions were performed with a PerkinElmer FlexDrop liquid dispenser. 10 mL
of 1.2 mM final assay concentration BCL6 BTB domain (amino acids
5–129 - C8Q, C67R, C84N)-mutant-AviT biotinylated and 10 mL of 10 nM final
assay concentration FP-labeled BCOR peptide (amino acids 498–514,
5-TAMRA-RSEIISTAPSSWVVPGP) were added to the assay-ready plates
containing the test compounds. Plates were then incubated at room temper-
ature for 1 hr and read on the BMG PHERAstar FS Multilabel Reader using the
instrument specifications for FP readout. Each plate contains negative
controls (BCOR peptide + assay buffer) and positive controls (BCL6 protein +
BCOR peptide).
BCL6-BCOR ULight Time-Resolved Fluorescence Resonance
Electron Transfer Assay
This assay was used to identify compounds that inhibit the binding of a BCOR
peptide to BCL6. Biotinylated BCL6 protein corresponding to BCL6 (amino
acids 5–129, with the following changes to the natural protein: C8Q, C67R,
C84N) was expressed in E. coli with a carboxy-terminal Avi tag (amino acid
sequence GLNDIFEAQKIEWHE). The BCOR ULight peptide uses a direct
carboxy-terminal ULight tag (PerkinElmer). The sequence of the peptide is
CRSEIISTAPSSWVVPGP, with the amino terminus acetylated and the ULight
tag attached to the cysteine. Compounds are dispensed onto assay plates
(Proxiplate-384 PLUS, white, PerkinElmer) using an Access Labcyte Worksta-
tion with the Labcyte Echo 55 3 from a DMSO solution. For the chosen highest
assay concentration of 100 mM, 150 nL of compound solution is transferred

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 R 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 R 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.

2870 Cell Reports 20, 2860–2875, September 19, 2017

 A

Farage, BI-3802 SU-DHL-4, BI-3802 OCI-Ly7, BI-3802 OCI-Ly-1, BI-3802

10000 10000 10 7 100000
Cumulative cell number

Cumulative cell number

Cumulative cell number

Cumulative cell number

10 6
10000
1000 1000
10 5

10 4
1000
3 µM
100 100 1 µM
10 3
100
0,3 µM
0,1 µM
10 10
10 2
0,04 µM
10 0,01 µM
10 1 no cpd
no cpd
1 1 10 0 1
0 5 10 15 20 0 5 10 15 0 5 10 15 20 0 5 10 15 20
Days Days Days Days

Farage, BI-3812 SU-DHL-4, BI-3812 OCI-Ly7, BI-3812 OCI-Ly-1, BI-3812

1000 10000 10 7 100000

Cumulative cell number

Cumulative cell number

Cumulative cell number

Cumulative cell number

10
10000
1000
10 5
100

10 4 1000 3 µM
100 1 µM
10 3
100
0,3 µM
10 0,1 µM
10
10 2 0,04 µM
10 0,01 µM
10 1 no cpd
no cpd
1 1 10 0 1
0 5 10 15 20 0 5 10 15 0 5 10 15 20 0 5 10 15 20
Days Days Days Days

Farage, BI-5273 Toledo, BI-3812 Toledo, BI-3802

10000 1000 1000
Cumulative cell number

Cumulative cell number

Cumulative cell number

1000

100 100

3 µM
100 1 µM
0,3 µM
10 10
0,1 µM
10 0,04 µM
0,01 µM
no cpd
no cpd
1 1 1
0 5 10 15 0 5 10 15 0 5 10 15
Days Days Days

B C

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).

Cell Reports 20, 2860–2875, September 19, 2017 2871

from a 10 mM DMSO compound stock solution. A series of 11 concentrations
is transferred for each compound at which each concentration is 5-fold lower
than the previous one. DMSO is added such that every well has a total of
150 nL compound solution. 5 mL of 7.5 nM BCL6 protein (final assay concen-
tration for ‘‘ULight’’ assay) or 5 mL of 1 nM BCL6 protein (final assay concen-
tration in ‘‘ULight LOW’’ assay; see below) in assay buffer (50 mM HEPES
[pH 7.3], 125 mM NaCl, 1 mM reduced glutathione [GSH], 0.01% Triton
X-100, 0.03% BSA) is added to the 150 nL of compounds. After 30 min incu-
bation time at room temperature, 10 mL of a mix containing BCOR ULight pep-
tide (100 nM final assay concentration) and Streptavidin-Europium (0.75 nM
final assay concentration) are added. Plates are kept at room temperature.
After 240 min incubation time, the time-resolved fluorescence resonance elec-
tron transfer (TR-FRET) signal is measured in a PerkinElmer Envision HTS
Multilabel Reader using the TR-FRET LANCE Ultra specs of PerkinElmer.
Each plate contains negative controls (diluted DMSO instead of test com-
pound; BCOR peptide and Streptavidin-Europium mix with BCL6 protein)
and positive controls (diluted DMSO instead of test compound; BCOR peptide
and Streptavidin-Europium mix without BCL6 protein). Negative and positive
control values are used for normalization. To measure compounds with IC50
values below 50 nM, the assay was modified to the ‘‘ULight LOW’’ assay
that uses less BCL6 protein in the assay (1 nM instead of 7.5 nM). ULight
and ULight LOW assay correlated well for BCL6 binders with an IC50
>50 nM (b = 0.79; r2 = 0.86) and were consequentially considered equitable
for such compounds.
BCL6 Degradation Assays
To quantify the effects of compounds on BCL6 degradation, SU-DHL-4 cells
were collected by centrifugation and re-suspended to 3 million cells per mL
in RPMI medium (ATCC #30-2001), 10% fetal bovine serum. 300,000 cells
were used per well of a 96-well plate in 100 mL medium. Compounds were
added to the cells at logarithmic dose series using the HP Digital Dispenser
D300 (Tecan), normalizing for added DMSO. After compound addition, cells
were incubated for 90 min at 37 C. Cells were collected by centrifugation,
washed once with PBS, and lysed in 25 mL lysis buffer (1% Triton, 350 mM
KCl, 10 mM Tris [pH 7.4]) supplemented with a phosphatase-protease inhibitor
cocktail (Thermo Scientific #1861281), 10 mM DTT, Benzonase 0.5 mL/mL
(Novagen #70746-10KU, 25 U/mL). BCL6 levels were analyzed using a Wes
capillary electrophoresis instrument (Proteinsimple) using BCL6 antibody
(4 mg/mL, 10 mL per lane rabbit, SIGMA #HPA004899) and GAPDH antibody
(1 mg/mL 10 mL per lane, rabbit, Abcam #ab8245) for normalization.
Proliferation Assays
For long-term proliferations assays, cells were passaged at a starting density
of 200,000 cells per mL in 1.5 mL in 24 well plates. Split rates were multiplied to
derive proliferation factors. See Supplemental Experimental Procedures for
details.
BCL6 Mutant and Chimeric Constructs
Expression constructs were generated by gene synthesis (GeneArt, Regens-
burg) in pDONR221 and expressed from pTREX-dest30 via transient transfec-
tion in HEK293 cells. See Supplemental Experimental Procedures for details.
RNA Sequence Analysis of Gene Expression
DMSO or compound from 1 mM stocks were added to cells at a density of
1 million cells per mL. For 7-day treatments, cells were split once after
3 days to 1 million cells per mL, and fresh compound or DMSO was added.
Cells were lysed in TRI lysis reagent (Sigma, T9424). Libraries from polyA-en-
riched RNA (protocols Quiagen, Illumina) were sequenced on an Illumina
HiSeq1500 using the paired-end protocol with 50 cycles and 25 million reads
in average. Gene and transcript quantification were performed using Cufflinks
version 2.0.2 (Trapnell et al., 2010). Differentially expressed genes between
treatment conditions and time points were computed using count or tran-
scripts per million (TPM) data and R version 3.3 coupled with the Bioconductor
(release 3.4) package DESeq2 (Love et al., 2014). All experiments were per-
formed in duplicates for estimating the variance and dispersion correction.
Only genes with more than 10 overlapping reads in at least one sample were
considered for analysis. Tables with differentially expressed genes were
2872 Cell Reports 20, 2860–2875, September 19, 2017
extended with additional gene-centric information retrieved via the Ensembl
BioMart service. If not otherwise stated in the manuscript, a default adjusted
p value of 0.01 (Benjamini-Hochberg correction) and an absolute fold-change
of 3 were used as a cutoff for all experiments. Gene-set enrichment analysis
was performed using the Bioconductor (release 3.4) package gage (Luo
et al., 2009).
qPCR Assays
For quantification of mRNA levels by PCR, 20,000 cells per well of a 96-well
plate were treated as indicated, collected by centrifugation at 250 g for
5 min, and washed and lysed in 50 mL lysis buffer using the fast lane cell multi-
plex kit (QIAGEN #216513). 2 mL of lysate was used per 25 mL PCR reaction.
Reaction mixes were generated by combining 12.5 mL QT MP RT PCR master
mix 2 3, 1.25 mL primer housekeeping gene 20 3, 1.25 mL primer target gene
20 3, 0.25 mL QT MP RT mix, and 7.75 mL water. Cycling conditions for RT-
qPCR were 20 min at 50 C, 15 min at 95 C, and 40 cycles of 15 s at 95 C +
45 s at 60 C. Cycle threshold numbers (Ct values) were determined for the
gene of interest and the control (GAPDH) PCR reactions. Differences in Ct
values for the target gene and GAPDH reactions (i.e., delta Ct values) were
calculated for each well. Data were transformed to a linear scale of mRNA
abundance by the following formula: Relative mRNA abundance = 2 (deltaCt).
The following primers were used, all from Applied Biosystems: PTPN6
Hs00169359_m1, RAPGEF1 Hs00178409_m1, BCL6 Hs00153368_m1, GAPDH
Hs03929097_g1, PRDM1 Hs00153357_m1, ATR Hs00992123_m1, CD69
Hs00934033_m1, CDKN1A (p21) Hs99999142_m1, TP53 Hs01034253_m1,
and CHST2 Hs01921028_s1.
Antibodies and Polyubiquitin Detection
For the detection of BCL6 in western blotting, the following antibodies were
used (target sequences in brackets): GeneTex #GTX1013 (center region of
human BCL6), Millipore #04-437 (C terminus), Raybio #168-10296 (C termi-
nus), Santa Cruz #sc-368 (C terminus of BCL6), Sigma #HPA004899 (center
region), Santa Cruz #sc-70414 (amino acids 3–484), and Santa Cruz #sc-
56625 (amino acids 3–484). For the detection of polyubiquitylated BCL6,
TUBE2 (tandem ubiquitin-binding entity) based on the ubiquitin-binding-asso-
ciated domain 1 from RAD23A coupled to horseradish peroxidase was used
(Life Sensors).
Immunoprecipitation
For immunoprecipitations, 4 million cells were lysed in 200 mL cell extrac-
tion buffer (ThermoFisher Scientific) containing 1 mM PMSF, 1 3 phospha-
tase/protease inhibitor cocktail (ThermoFisher Scientific), and 50 mM
iodoacetamide. Lysates were cleared by centrifugation and 150 mL was
precipitated with 1 mg BCL6 antibody (Santa Cruz #sc-368) and 50 mL pro-
tein G Dynabeads (ThermoFisher Scientific), washed 6 times with PBS, and
analyzed by western blotting using BCL6 antibody (SIGMA HPA004899) or
TUBEs.
Chemoproteomics
Preparation of Affinity Matrix
The linkable BI-3802 analog 5b was incubated with NHS Mag Sepharose (GE
Healthcare) for 20 hr in 1% triethylamine in DMSO at a ligand density of 10%,
and free sites were quenched with aminoethanol.
Preparation of Cell Lysate
Pellets of Farage cells were suspended in lysis buffer (20 mM HEPES [pH 7.5],
350 mM KCl, 1% Triton X-100), supplemented with protease inhibitors
(Roche), and rotated for 20 min. Lysates were cleared at 30,000 3 g.
Chemoaffinity Pull-Down
Magnetic particle processing was parallelized in a 96-well format using a
magnetic particle processor (KingFisher, ThermoFisher Scientific). Per reac-
tion, 1 mL affinity matrix was washed with lysis buffer and incubated with
lysate in 20 mM HEPES (pH 7.5), 350 mM KCl, and 0.8% Triton X-100 for
2 hr at 4 C.
Sample Preparation
Proteins bound to the washed beads were proteolytically eluted with Lys-C
(Wako Chemicals) in the presence of 5 mM Tris(2-carboxyethyl)phosphine
(TCEP), 50 mM triethanolamine (pH 8.5) in water, and 2.5 fmol/mL digested
BSA for 2 hr at 30 C. Thiols were reduced with 5 mM iodoacetamide, and
peptides were further digested with Trypsin (ThermoFisher Scientific) in
50 mM triethanolamine (pH 8.5) for 15 hr at 37 C. Reactions were acidified
with trifluoroacetic acid, purified on Sep-Pak tC18 mElution plates (Waters),
and dried down to completion in a vacuum centrifuge.
Liquid Chromatography-MS Analysis
NanoLC-MS/MS was performed on a RSLC Nano (ThermoFisher Scientific)
interfaced to a Q Exactive Plus mass spectrometer (ThermoFisher Scientific).
The LC system was equipped with 2 cm L 3 100 mm ID pre-column (PepMap
C18, 5 mm particle size, ThermoFisher Scientific) and a 25 cm L 3 75 mm ID
(PepMap C18, 2 mm particle size, ThermoFisher Scientific). Samples were
injected onto the pre-column at 20 mL/min with 0.05% trifluoroacetic acid
(TFA) in water and separated at 300 nL/min at 35 C using a gradient of 2% sol-
vent B (80% acetonitrile, 0.1% formic acid in water) in solvent A (0.1% formic
acid in water) to 35% solvent B in solvent A in 60 min, followed by a gradient to
90% solvent B in solvent A in 5 min. The mass spectrometer was set to record
MS spectra between 400 and 1400 m/z with an automatic gain control (AGC)
target value of 3e6 and a resolving power of 70,000. It was set to fragment the
top 5 most intense peaks with a charge >1+ with 20 s dynamic exclusion time.
MS/MS spectra were recorded with an AGC target value of 1e5, an intensity
threshold of 1e5, a maximal accumulation time of 200 ms, and a resolving
power of 17,000.
Data Analysis
Label-free quantification and peak list generation was performed using
Progenesis for Proteomics (version 3.0, Waters). Runs were normalized to
BSA peptides with a maximal coefficient of variation (CV) <10%. Features
with <3 isotopes or charge >4+ or <2+, and label-free intensities with a
maximal CV of 2 assay replicates >25% throughout all conditions were dis-
qualified. MS/MS peak number limitation and peak de-isotoping were enabled
for peak list generation. The peak list was searched against the UniProtKB
Human Reference Proteome using Mascot (version 2.5, Matrix Sciences)
with 5 ppm precursor and 20 ppm fragment ion mass tolerances, carbamido-
methylation, and protein N-terminal acetylation as variable modifications.
Peptide-spectrum matches (PSMs) were filtered to a significance
of < 0.01% and an ions score >20, and ‘‘bold red’’ was required for extensible
markup language (XML) export. These settings resulted in a PSM-level false-
discovery rate <1%. Data were visualized using Spotfire (version 6.5, TIBCO
Software).
Statistical Analysis
Averages and SDs from n R 3 samples were calculated, and averages were
compared using unpaired t tests. p values < 0.05 were considered to indicate
statistical significance. Concentrations of half-maximal effect were calculated
from triplicate measurements using four-parameter non-linear regression.
Statistical analysis for RNA sequencing (RNA-seq), SPR, and chemoproteomic
analysis is described in the respective section.
ACCESSION NUMBERS
All crystal structures have been deposited in the Protein Data Bank (PDB):
5MW6 (structure of BCL6 with compound 1), 5MWD (structure of BCL6 with
compound 2), and 5MW2 (structure of BCL6 with BI-3802). The accession
number for the data reported in this paper is GenBank: GSE94099.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and six tables and can be found with this article online at
http://dx.doi.org/10.1016/j.celrep.2017.08.081.
AUTHOR CONTRIBUTIONS
N. Kerres, S. Steurer, U.R., S.L., and D.M. directed chemical research. G.B.,
D.K., A.W.-P., G.F., and A.Z. provided X-ray crystallography analyses. C.D.,
P.G., C.P., and O.P. performed the synthesis of the small molecules. B.H.,
M.C., D.R.L., and X.Z. contributed to the structure-activity relationship under-
standing and design of chemical analogs at the very beginning of the project.
T. Gerstberger, P.E., and M.K. developed and performed biochemical assays
to measure inhibition of BCL6 by small molecules. D.K. did screens for small
molecules binding to BCL6. N.M. performed mass spectrometry. S.W. con-
ducted SPR assays. R.E.I. and J.R.E. conducted HDX experiments, E.H. con-
ducted LUMIER assays, and T. Gmaschitz conducted LUMIER assays, degra-
dation assays, immunoprecipitations, and qPCRs. D.T., A.S., and B.F.
performed proliferation assays. E.T. performed degradation assays. H.B.,
M.M., K.R., and D.S. were involved in biophysical characterization of small
molecules. H.K. and K. Mu €ck performed differential scanning fluorimetry.
K.M. tested formulations of BI-3802. R.S., S. Schlager, and L.L. performed
qPCR analyses. D.T., C.S., R.V., and N.S. performed the RNA sequencing.
D.G. and T.V. conducted bioinformatic analyses. N. Kerres and D.M. super-
vised the chemical research. N. Kraut, M.P., P.E., D.R., and M.K. supervised
biological research. N. Kerres and M.K. wrote the manuscript.
ACKNOWLEDGMENTS
We would like to thank Teresa Adacker, Sophia Blake, Margit Bauer, Xiaoling
Cockcroft, Doug R. Davies (Beryllium Discovery), Gerlinde Flotzinger, Julian
Fuchs, Gabriele Glendinning, Gerhard Gmaschitz, Elisabeth Grondal,
Wolfgang Hela, Karin Hofbauer, Gudrun Illibauer, Elisabeth Jirkovsky, Thomas
Karner, Martina Kohla, Roland Kousek, Susanne Mayer, David Myszka
(Biosensor Tools), Sabine Olt, Martin Perlinger, Carlos R. Ramirez-Santa
Cruz, Gorana Sijan, Andreas Schrenk, Elisabeth Traxler, Anika Weiss, Patrick
Werni, and Jasmin Zimmer for excellent project support and Heribert Arnhof
for advice. All authors except M.C., R.E.I., J.R.E., B.H., D.R.L., and X.Z.
were or are employees of Boehringer Ingelheim. S. Schlager was supported
by a Fempower Grant (1433765) of the Wirschaftsagentur Wien.
Received: March 10, 2017
Revised: June 29, 2017
Accepted: August 24, 2017
Published: September 19, 2017
REFERENCES
Ahmad, K.F., Engel, C.K., and Prive, G.G. (1998). Crystal structure of the BTB
domain from PLZF. Proc Natl Acad Sci U S A 95, 12123–12128.
Ahmad, K.F., Melnick, A., Lax, S., Bouchard, D., Liu, J., Kiang, C.L., Mayer, S.,
Takahashi, S., Licht, J.D., and Privé, G.G. (2003). Mechanism of SMRT core-
pressor recruitment by the BCL6 BTB domain. Mol. Cell 12, 1551–1564.
Bardwell, V.J., and Treisman, R. (1994). The POZ domain: a conserved protein-
protein interaction motif. Genes Dev. 8, 1664–1677.
Barrios-Rodiles, M., Brown, K.R., Ozdamar, B., Bose, R., Liu, Z., Donovan,
R.S., Shinjo, F., Liu, Y., Dembowy, J., Taylor, I.W., et al. (2005). High-
throughput mapping of a dynamic signaling network in mammalian cells. Sci-
ence 307, 1621–1625.
Basso, K., and Dalla-Favera, R. (2012). Roles of BCL6 in normal and trans-
formed germinal center B cells. Immunol. Rev. 247, 172–183.
Basso, K., and Dalla-Favera, R. (2015). Germinal centres and B cell lymphoma-
genesis. Nat. Rev. Immunol. 15, 172–184.
Basso, K., Saito, M., Sumazin, P., Margolin, A.A., Wang, K., Lim, W.K., Kita-
gawa, Y., Schneider, C., Alvarez, M.J., Califano, A., and Dalla-Favera, R.
(2010). Integrated biochemical and computational approach identifies BCL6
direct target genes controlling multiple pathways in normal germinal center
B cells. Blood 115, 975–984.
Blasche, S., and Koegl, M. (2013). Analysis of protein-protein interactions
using LUMIER assays. Methods Mol. Biol. 1064, 17–27.
Cardenas, M.G., Yu, W., Beguelin, W., Teater, M.R., Geng, H., Goldstein, R.L.,
Oswald, E., Hatzi, K., Yang, S.N., Cohen, J., et al. (2016). Rationally designed
BCL6 inhibitors target activated B cell diffuse large B cell lymphoma. J. Clin.
Invest. 126, 3351–3362.
Cattoretti, G., Pasqualucci, L., Ballon, G., Tam, W., Nandula, S.V., Shen, Q.,
Mo, T., Murty, V.V., and Dalla-Favera, R. (2005). Deregulated BCL6 expression
Cell Reports 20, 2860–2875, September 19, 2017 2873
recapitulates the pathogenesis of human diffuse large B cell lymphomas in
mice. Cancer Cell 7, 445–455.
Cerchietti, L.C., Ghetu, A.F., Zhu, X., Da Silva, G.F., Zhong, S., Matthews, M.,
Bunting, K.L., Polo, J.M., Farès, C., Arrowsmith, C.H., et al. (2010). A small-
molecule inhibitor of BCL6 kills DLBCL cells in vitro and in vivo. Cancer Cell
17, 400–411.
Chaharbakhshi, E., and Jemc, J.C. (2016). Broad-complex, tramtrack, and
bric-à-brac (BTB) proteins: Critical regulators of development. Genesis 54,
505–518.
Ci, W., Polo, J.M., Cerchietti, L., Shaknovich, R., Wang, L., Yang, S.N., Ye, K.,
Farinha, P., Horsman, D.E., Gascoyne, R.D., et al. (2009). The BCL6 transcrip-
tional program features repression of multiple oncogenes in primary B cells
and is deregulated in DLBCL. Blood 113, 5536–5548.
Cleasby, A., Yon, J., Day, P.J., Richardson, C., Tickle, I.J., Williams, P.A., Call-
ahan, J.F., Carr, R., Concha, N., Kerns, J.K., et al. (2014). Structure of the BTB
domain of Keap1 and its interaction with the triterpenoid antagonist CDDO.
PLoS ONE 9, e98896.
Dent, A.L., Shaffer, A.L., Yu, X., Allman, D., and Staudt, L.M. (1997). Control of
inflammation, cytokine expression, and germinal center formation by BCL-6.
Science 276, 589–592.
Di Leo, A., Jerusalem, G., Petruzelka, L., Torres, R., Bondarenko, I.N., Khasa-
nov, R., Verhoeven, D., Pedrini, J.L., Smirnova, I., Lichinitser, M.R., et al.
(2010). Results of the CONFIRM phase III trial comparing fulvestrant 250 mg
with fulvestrant 500 mg in postmenopausal women with estrogen receptor-
positive advanced breast cancer. J. Clin. Oncol. 28, 4594–4600.
Duan, S., Cermak, L., Pagan, J.K., Rossi, M., Martinengo, C., di Celle, P.F.,
Chapuy, B., Shipp, M., Chiarle, R., and Pagano, M. (2012). FBXO11 targets
BCL6 for degradation and is inactivated in diffuse large B-cell lymphomas. Na-
ture 481, 90–93.
Engen, J.R. (2009). Analysis of protein conformation and dynamics by
hydrogen/deuterium exchange MS. Anal. Chem. 81, 7870–7875.
Furukawa, M., He, Y.J., Borchers, C., and Xiong, Y. (2003). Targeting of protein
ubiquitination by BTB-Cullin 3-Roc1 ubiquitin ligases. Nat. Cell Biol. 5, 1001–
1007.
Geyer, R., Wee, S., Anderson, S., Yates, J., and Wolf, D.A. (2003). BTB/POZ
domain proteins are putative substrate adaptors for cullin 3 ubiquitin ligases.
Mol. Cell 12, 783–790.
Ghetu, A.F., Corcoran, C.M., Cerchietti, L., Bardwell, V.J., Melnick, A., and
Privé, G.G. (2008). Structure of a BCOR corepressor peptide in complex
with the BCL6 BTB domain dimer. Mol. Cell 29, 384–391.
Hatzi, K., and Melnick, A. (2014). Breaking bad in the germinal center: how
deregulation of BCL6 contributes to lymphomagenesis. Trends Mol. Med.
20, 343–352.
Huang, C., Hatzi, K., and Melnick, A. (2013). Lineage-specific functions of
Bcl-6 in immunity and inflammation are mediated by distinct biochemical
mechanisms. Nat. Immunol. 14, 380–388.
Huang, C., Gonzalez, D.G., Cote, C.M., Jiang, Y., Hatzi, K., Teater, M., Dai, K.,
Hla, T., Haberman, A.M., and Melnick, A. (2014). The BCL6 RD2 domain gov-
erns commitment of activated B cells to form germinal centers. Cell Rep. 8,
1497–1508.
Huerta, C., Jiang, X., Trevino, I., Bender, C.F., Ferguson, D.A., Probst, B.,
Swinger, K.K., Stoll, V.S., Thomas, P.J., Dulubova, I., et al. (2016). Character-
ization of novel small-molecule NRF2 activators: Structural and biochemical
validation of stereospecific KEAP1 binding. Biochim. Biophys. Acta 1860 (11
Pt A), 2537–2552.
Ji, A.X., Chu, A., Nielsen, T.K., Benlekbir, S., Rubinstein, J.L., and Privé, G.G.
(2016). Structural Insights into KCTD Protein Assembly and Cullin3 Recogni-
tion. J. Mol. Biol. 428, 92–107.
Jordan, V.C. (2003). Tamoxifen: a most unlikely pioneering medicine. Nat. Rev.
Drug Discov. 2, 205–213.
Kamada, Y., Sakai, N., Sogabe, S., Ida, K., Oki, H., Sakamoto, K., Lane, W.,
Snell, G., Iida, M., Imaeda, Y., et al. (2017). Discovery of a B-Cell Lymphoma
2874 Cell Reports 20, 2860–2875, September 19, 2017
6 Protein-Protein Interaction Inhibitor by a Biophysics-Driven Fragment-Based
Approach. J. Med. Chem. 60, 4358–4368.
Knutson, S.K., Wigle, T.J., Warholic, N.M., Sneeringer, C.J., Allain, C.J., Klaus,
C.R., Sacks, J.D., Raimondi, A., Majer, C.R., Song, J., et al. (2012). A selective
inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells.
Nat. Chem. Biol. 8, 890–896.
Li, X., Peng, H., Schultz, D.C., Lopez-Guisa, J.M., Rauscher, F.J., 3rd, and
Marmorstein, R. (1999). Structure-function studies of the BTB/POZ transcrip-
tional repression domain from the promyelocytic leukemia zinc finger onco-
protein. Cancer Res. 59, 5275–5282.
Liu, Z., Song, F., Ma, Z.L., Xiong, Q., Wang, J., Guo, D., and Sun, G. (2016).
Bivalent Copper Ions Promote Fibrillar Aggregation of KCTD1 and Induce
Cytotoxicity. Sci. Rep. 6, 32658.
Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold
change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550.
Lu, J., Qian, Y., Altieri, M., Dong, H., Wang, J., Raina, K., Hines, J., Winkler,
J.D., Crew, A.P., Coleman, K., and Crews, C.M. (2015). Hijacking the
E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4. Chem. Biol. 22,
755–763.
Luo, W., Friedman, M.S., Shedden, K., Hankenson, K.D., and Woolf, P.J.
(2009). GAGE: generally applicable gene set enrichment for pathway analysis.
BMC Bioinformatics 10, 161.
McCoull, W., Abrams, R.D., Anderson, E., Blades, K., Barton, P., Box, M.,
Burgess, J., Byth, K., Cao, Q., Chuaqui, C., et al. (2017). Discovery of Pyra-
zolo[1,5-a]pyrimidine B-Cell Lymphoma 6 (BCL6) Binders and Optimization
to High Affinity Macrocyclic Inhibitors. J. Med. Chem. 60, 4386–4402.
Mei, S., Qin, Q., Wu, Q., Sun, H., Zheng, R., Zang, C., Zhu, M., Wu, J., Shi, X.,
Taing, L., et al. (2017). Cistrome Data Browser: a data portal for ChIP-Seq and
chromatin accessibility data in human and mouse. Nucleic Acids Res. 45 (D1),
D658–D662.
Mesin, L., Ersching, J., and Victora, G.D. (2016). Germinal Center B Cell Dy-
namics. Immunity 45, 471–482.
Migliazza, A., Martinotti, S., Chen, W., Fusco, C., Ye, B.H., Knowles, D.M.,
Offit, K., Chaganti, R.S., and Dalla-Favera, R. (1995). Frequent somatic hyper-
mutation of the 50 noncoding region of the BCL6 gene in B-cell lymphoma.
Proc. Natl. Acad. Sci. USA 92, 12520–12524.
Nance, J.P., Bélanger, S., Johnston, R.J., Hu, J.K., Takemori, T., and Crotty, S.
(2015). Bcl6 middle domain repressor function is required for T follicular helper
cell differentiation and utilizes the corepressor MTA3. Proc. Natl. Acad. Sci.
USA 112, 13324–13329.
Niu, H., Ye, B.H., and Dalla-Favera, R. (1998). Antigen receptor signaling in-
duces MAP kinase-mediated phosphorylation and degradation of the BCL-6
transcription factor. Genes Dev. 12, 1953–1961.
Osborne, C.K., Wakeling, A., and Nicholson, R.I. (2004). Fulvestrant: an oestro-
gen receptor antagonist with a novel mechanism of action. Br. J. Cancer 90
(Suppl 1), S2–S6.
Pasqualucci, L. (2013). The genetic basis of diffuse large B-cell lymphoma.
Curr. Opin. Hematol. 20, 336–344.
Pasqualucci, L., Migliazza, A., Basso, K., Houldsworth, J., Chaganti, R.S., and
Dalla-Favera, R. (2003). Mutations of the BCL6 proto-oncogene disrupt its
negative autoregulation in diffuse large B-cell lymphoma. Blood 101, 2914–
2923.
Pasqualucci, L., Dominguez-Sola, D., Chiarenza, A., Fabbri, G., Grunn, A., Tri-
fonov, V., Kasper, L.H., Lerach, S., Tang, H., Ma, J., et al. (2011). Inactivating
mutations of acetyltransferase genes in B-cell lymphoma. Nature 471,
189–195.
Perez-Torrado, R., Yamada, D., and Defossez, P.A. (2006). Born to bind: the
BTB protein-protein interaction domain. BioEssays 28, 1194–1202.
Phan, R.T., Saito, M., Kitagawa, Y., Means, A.R., and Dalla-Favera, R. (2007).
Genotoxic stress regulates expression of the proto-oncogene Bcl6 in germinal
center B cells. Nat. Immunol. 8, 1132–1139.
Raina, K., Lu, J., Qian, Y., Altieri, M., Gordon, D., Rossi, A.M., Wang, J., Chen,
X., Dong, H., Siu, K., et al. (2016). PROTAC-induced BET protein degradation
as a therapy for castration-resistant prostate cancer. Proc. Natl. Acad. Sci.
USA 113, 7124–7129.
Schneider, C., Kon, N., Amadori, L., Shen, Q., Schwartz, F.H., Tischler, B.,
Bossennec, M., Dominguez-Sola, D., Bhagat, G., Gu, W., et al. (2016).
FBXO11 inactivation leads to abnormal germinal-center formation and lym-
phoproliferative disease. Blood 128, 660–666.
Siggs, O.M., and Beutler, B. (2012). The BTB-ZF transcription factors. Cell
Cycle 11, 3358–3369.
Soucy, T.A., Smith, P.G., Milhollen, M.A., Berger, A.J., Gavin, J.M., Adhikari,
S., Brownell, J.E., Burke, K.E., Cardin, D.P., Critchley, S., et al. (2009). An in-
hibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature
458, 732–736.
Stead, M.A., Trinh, C.H., Garnett, J.A., Carr, S.B., Baron, A.J., Edwards, T.A.,
and Wright, S.C. (2007). A beta-sheet interaction interface directs the tetra-
merisation of the Miz-1 POZ domain. J. Mol. Biol. 373, 820–826.
Toure, M., and Crews, C.M. (2016). Small-Molecule PROTACS: New Ap-
proaches to Protein Degradation. Angew. Chem. Int. Ed. Engl. 55, 1966–1973.
Trapnell, C., Williams, B.A., Pertea, G., Mortazavi, A., Kwan, G., van Baren,
M.J., Salzberg, S.L., Wold, B.J., and Pachter, L. (2010). Transcript assembly
and quantification by RNA-Seq reveals unannotated transcripts and isoform
switching during cell differentiation. Nat. Biotechnol. 28, 511–515.
Winter, G.E., Buckley, D.L., Paulk, J., Roberts, J.M., Souza, A., Dhe-Paganon,
S., and Bradner, J.E. (2015). DRUG DEVELOPMENT. Phthalimide conjugation
as a strategy for in vivo target protein degradation. Science 348, 1376–1381.
Xu, L., Wei, Y., Reboul, J., Vaglio, P., Shin, T.H., Vidal, M., Elledge, S.J., and
Harper, J.W. (2003). BTB proteins are substrate-specific adaptors in an
SCF-like modular ubiquitin ligase containing CUL-3. Nature 425, 316–321.
Ye, B.H., Lista, F., Lo Coco, F., Knowles, D.M., Offit, K., Chaganti, R.S., and
Dalla-Favera, R. (1993). Alterations of a zinc finger-encoding gene, BCL-6, in
diffuse large-cell lymphoma. Science 262, 747–750.
Ye, B.H., Cattoretti, G., Shen, Q., Zhang, J., Hawe, N., de Waard, R., Leung, C.,
Nouri-Shirazi, M., Orazi, A., Chaganti, R.S., et al. (1997). The BCL-6 proto-
oncogene controls germinal-centre formation and Th2-type inflammation.
Nat. Genet. 16, 161–170.
Ying, C.Y., Dominguez-Sola, D., Fabi, M., Lorenz, I.C., Hussein, S., Bansal, M.,
Califano, A., Pasqualucci, L., Basso, K., and Dalla-Favera, R. (2013). MEF2B
mutations lead to deregulated expression of the oncogene BCL6 in diffuse
large B cell lymphoma. Nat. Immunol. 14, 1084–1092.
Zollman, S., Godt, D., Privé, G.G., Couderc, J.L., and Laski, F.A. (1994). The
BTB domain, found primarily in zinc finger proteins, defines an evolutionarily
conserved family that includes several developmentally regulated genes in
Drosophila. Proc. Natl. Acad. Sci. USA 91, 10717–10721.
Cell Reports 20, 2860–2875, September 19, 2017 2875