Proteasome inhibitors suppress MYB oncogenic activity in a
p300-dependent manner
Maria V. Yusenko a,1
, Abhiruchi Biyanee a,1
, Mattias K. Andersson b
, Silke Radetzki c
Jens P. von Kries c
, Goran ¨ Stenman b
, Karl-Heinz Klempnauer a,*
a Institute for Biochemistry, Westfalische-Wilhelms-Universit ¨ at, ¨ Münster, Germany b Sahlgrenska Cancer Center, Department of Pathology, University of Gothenburg, Gothenburg, Sweden c Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany
ARTICLE INFO
Keywords:
MYB
Inhibitor
Myeloid leukemia
Adenoid cystic carcinoma
Proteasome inhibitor
ABSTRACT
Studies of the role of MYB in human malignancies have highlighted MYB as a potential drug target for acute
myeloid leukemia (AML) and adenoid cystic carcinoma (ACC). Although transcription factors are often
considered un-druggable, recent work has demonstrated successful targeting of MYB by low molecular weight
compounds. This has fueled the notion that inhibition of MYB has potential as a therapeutic approach against
MYB-driven malignancies. Here, we have used a MYB reporter cell line to screen a library of FDA-approved drugs
for novel MYB inhibitors. We demonstrate that proteasome inhibitors have significant MYB-inhibitory activity,
prompting us to characterize the proteasome inhibitor oprozomib in more detail. Oprozomib was shown to
interfere with the ability of the co-activator p300 to stimulate MYB activity and to exert anti-proliferative effects
on human AML and ACC cells. Overall, our work demonstrated suppression of oncogenic MYB activity as a novel
result of proteasome inhibition.
1. Introduction
Recent work has highlighted the transcription factor MYB as a novel
drug target for treatment of malignancies depending on elevated MYB
expression, such as acute myeloid leukemia (AML), or driven by rear￾ranged MYB as demonstrated for adenoid cystic carcinoma (ACC) [1–5].
AML is a common and aggressive type of leukemia of children and adults
with a poor prognosis, especially for elderly patients [6]. MYB plays a
central role in AML as a master regulator of oncogenic transcriptional
programs essential for self-renewal and leukemic maintenance [7,8].
MYB has also been implicated in the development of T-cell acute
lymphoblastic leukemia (T-ALL), due to duplications or translocations of
MYB or to point mutations generating MYB-responsive super-enhancers
upstream of the TAL1 or LMO2 oncogenes [9–13]. ACC is a rare,
slow-growing but aggressive cancer that arises in salivary glands or
other anatomical sites [14]. In its advanced stage, the disease is gener￾ally incurable due to the lack of effective systemic therapies. Recurrent
translocations resulting in MYB-NFIB gene fusions, or variants thereof,
have been identified in a large percentage of cases as key drivers in ACC
[4,5,15]. Recent evidence has suggested that the translocations re-direct
MYB-dependent super-enhancers towards the MYB gene, thereby
generating a feedback loop that leads to high expression of MYB fusion
proteins [16].
MYB was initially discovered as the cellular progenitor of the v-myb
oncogene of avian myeloblastosis virus [17]. MYB is highly expressed in
immature hematopoietic progenitor cells where it plays crucial roles in
the development and homeostasis of the hematopoietic system [18]. As
a transcription factor, MYB is highly dependent on cooperation with the
transcriptional co-activator p300. MYB interacts with p300 via a
conserved LXXLL-motif present in the MYB transactivation domain and
the KIX-domain of p300 [19]. Mutation of Leu-302 or Met-303 within or
immediately adjacent to the LXXLL-motif disturb the interaction with
p300 and weaken the activity of MYB, thus establishing the importance
of the LXXLL-motif for MYB activity [20–22]. In recent years, MYB has
gained attention as a potential drug target [1,3,4,23]. AML cells were
shown to depend on higher levels of MYB than normal hematopoietic
progenitor cells, making them more vulnerable to MYB inhibition than
their normal counterparts [7,24,25]. Recent studies have pioneered
targeting of MYB by low-molecular weight compounds that disrupt the
* Corresponding author. Institute for Biochemistry, Westfalische-Wilhelms-Universit ¨ ¨
at, D-48149, Muenster, Germany.
E-mail address: [email protected] (K.-H. Klempnauer). 1 Both authors contributed equally to the work.
Contents lists available at ScienceDirect
Cancer Letters
journal homepage: www.elsevier.com/locate/canlet

https://doi.org/10.1016/j.canlet.2021.07.010

Received 22 April 2021; Received in revised form 18 June 2021; Accepted 6 July 2021
Cancer Letters 520 (2021) 132–142
133
MYB/p300 interaction, showing for the first time that MYB is a drug￾gable transcription factor [26–30]. Importantly, these studies have
confirmed that blocking MYB activity is effective against AML in an in
vivo mouse model. Furthermore, inhibition of MYB was found to be
effective in inhibiting the proliferation of ACC cells in vitro and in mouse
models [4,31,32]. Overall, these studies suggest that pharmacological
inhibition of MYB may have potential as a novel therapeutic strategy
against MYB-dependent malignancies.
To identify novel MYB inhibitors we have screened a library of FDA￾approved drugs for inhibitory activity, using a recently established MYB
reporter cell line [33]. Here, we show that proteasome inhibitors are
highly potent MYB-inhibitory agents that suppress MYB oncogenic ac￾tivity in a p300-dependent manner.
2. Materials and methods
2.1. Cells
Hek293T, HepG2, U2OS and HeLa are non-hematopoietic adherent
human cell lines. NB4, HL60, U937 and THP1 are human myeloid leu￾kemia cell lines. All cell lines were originally obtained from ATCC and
were free of mycoplasma contamination. HL60 cells expressing a C￾terminally truncated MYB (MYB-CT3) were generated by lentiviral
infection as described before [33]. Control HL60 cells were transfected
with an “empty” lentivirus. Patient-derived ACC cells were cultured as
described [4]. Cytospin slides were stained with May-Grunwald Giemsa.
2.2. Library screening
The HEK-MYB-Luc and HEK-Luc reporter cell lines have been
described before [33]. Screening of the Selleck FDA-approved Drug Li￾brary (SelleckChem) and the LOPACR1280 library (Sigma-Aldrich),
covering together more than 4000 FDA-approved drugs and compounds
with annotated activities, was performed in 384-well plates by the
screening unit of the Leibniz-Forschungsinstitut für Molekulare Phar￾makologie (FMP). Compounds were added at a final concentration of 10
μM to wells containing HEK-MYB-Luc cells pretreated with doxycyclin.
The luciferase-based reporter signals were detected with the Steady-Glo
luciferase kit (Promega) after 24 h of incubation, using a TECAN
microplate reader. Data were analyzed based on the Z score (the number
of standard deviations, SDs, a measured signal intensity is above the
mean). Selected compounds were re-tested in triplicates in
HEK-MYB-Luc and HEK-Luc cells at 10 μM concentration. Candidate
compounds were then subjected to IC50 determinations with compound
concentrations ranging from 20 to 0.01 μM.
2.3. Expression vectors
Expression vectors for MYB, MYB-2KR, MYB-CT3 and Gal4-CT3 have
been described [27,34]. M303V and L302A mutant derivatives of
MYB-CT3 were obtained by site-directed mutagenesis. The p300
expression vector was originally obtained from R. Eckner [35]. A
GAL4-p300(1853–2414) expression vector was generated by fusing the
coding region for amino acids 1853 to 2414 of p300 with the coding
region of the Gal4 DNA-binding domain, using appropriate restriction
sites. The MYB- and Gal4-inducible luciferase reporter plasmids
pGL4-5xMRE(GG)-Myc (containing 5 tandem copies of a Myb binding
site upstream of a core promoter) and pG5E4-38Luc (containing 5 copies
of the Gal4 binding site upstream of the minimal adenovirus E4 pro￾moter have been described [36,37]. The C/EBP-dependent reporter
plasmid pmim3mim-Luc has been described [38]. The GFI1 luciferase
reporter plasmid was generated by PCR-amplifying the promoter region
with primers 5′
-ATATATGGTACCACCGCGCTAGGAGAGTTTTC-3′ and
5′
-ATATATCTCGAGCGCCAGTCAATCTGTGTCCT-3′ and cloning it be￾tween the KpnI and XhoI sites of pGL3-Luc.
2.4. Electrophoretic mobility shift assays (EMSA)
In vitro DNA-binding assays were carried out as described before,
using a synthetic double-stranded oligonucleotide containing a high
affinity MYB binding site [38]. Nuclear extracts were prepared from
non-transfected HEK293T cells and from HEK293T cells transfected
with expression vector for MYB-CT3 and incubated for 16 h in the
absence or presence of relevant concentrations of oprozomib.
2.5. Transfections and co-immunoprecipitation
Transfection of HEK293T cells by calcium-phosphate co-precipita￾tion and reporter assays were performed as previously described [39].
Co-immunoprecipitation was performed using GFP-trap beads (Chro￾motec, München) containing a covalently bound nanobody with high
affinity to GFP. Cells were lysed in ELB buffer (50 mM Tris/HCl pH 7,5;
120 mM NaCl; 20 mM NaF; 1 mM EDTA; 6 mM EGTA; 15 mM sodium
pyrophosphate; 1 mM phenylmethylsulfonylfluoride; 0,2% NP-40 and a
protease inhibitor mix containing Aprotinin, Leupeptin and Pepstatin).
After incubation on ice for 15 min, lysates were centrifuged at 14000×g
for 15 min, and the supernatant was used as total cell extract. Aliquots of
cell extracts were then incubated with GFP-trap beads for 3 h at 4 ◦C.
Beads were washed 4 times with ELB buffer. Bound proteins and input
samples were analyzed by Western blotting with appropriate antibodies.
2.6. Quantitative real-time PCR
RT-PCR of MYC, KIT, GFI1, and ACTB mRNA expression was per￾formed as described [32]. All experiments were conducted with at least
three biological replicates and the following primers:
MYC: 5′
-GCCGATCAGCTGGAGATGA-3′ and 5′
-GTCGTCAGGATCG￾CAGATGAAG-3’;
KIT: 5′
-TGATTTTCCTGGATGGATGG-3′ and 5′
TGGGATTTTCTCTGCGTTCT-3’;
GFI1: 5′
-GCTCGGAGTTTGAGGACTTC-3′ and 5′
-ATGGGCA￾CATTGACTTCTCC-3’;
ACTB: 5′
-AGAGCTACGAGCTGCCTGAC-3′ and 5′
AGCACTGTGTTGGCGTACAG-3’
2.7. Flow cytometry
Approximately 106 HL60 cells were cultured for 2 days in RPMI 1640
medium containing the desired concentration of oprozomib. Control
cells were incubated without compound. The cells were then analyzed
by flow cytometry for CD11b expression by using PE/Cy7-labeled anti￾human CD11b (ICRF44, BioLegend) and a FC 500 Cytometer (Beckman
Coulter). Apoptotic and necrotic cells were determined by double￾staining with FITC-annexin-V (BioLegend) and propidium iodide (PI).
CXP software (Beckman Coulter) was used for subsequent analysis.
Abbreviations
EMSA Electrophoretic mobility shift assay
Gal4 Galactose-responsive transcription factor GAL4
GFI1 growth factor independent 1 transcriptional repressor
KIT KIT proto-oncogene, receptor tyrosine kinase
MYC MYC proto-oncogene, bHLH transcription factor
MYB MYB proto-oncogene, transcription factor
MYBL1 MYB proto-oncogene like 1
LMO2 LIM domain only 2
TAL1 TAL bHLH transcription factor 1, erythroid
differentiation factor
2.8. Proliferation, apoptosis, and sphere assays of ACC cells
Cell viability of hematipoietic and control cell lines was determined
by MTT assays. Cells were incubated with compounds for 24 h, followed
by addition of MTT solution (Millipore Corp., USA), incubation for 4 h
and measuring the absorbance at 495 nm with a microplate photometer
(MPP 4008, Mikrotek). For proliferation assays of ACC cells, 4,000 cells
were seeded per well in black 96-well plates (BD) and treated the
following day with different concentrations of oprozomib or DMSO as
control. Cells were assayed 72 h later with the Alamar blue reagent
(Thermo Fisher ScientiFIc) according to the manufacturer’s instructions.
For apoptosis assays, 8,000 ACC cells were seeded in white-walled 96-
well plates (BD) and treated the next day with either DMSO, 250 or
1000 nM oprozomib for 24 h. Apoptosis was assayed with the Caspase￾Glo 3/7 reagent (Promega). For sphere assays, 50,000 ACC cells were
seeded in Nunclon delta 6-well plates (Thermo Fisher Scientific) pre￾coated with PolyHEMA (12 g/l in 95% ethanol; Sigma-Aldrich) and
cultured in the presence of 100 nM oprozomib (with DMSO as a control)
for 10 days. MTT (0.25 mg/ml, Sigma-Aldrich) was added to wells for 1
h at 37 ◦C, and spheres were quantified with the GelCount colony
counter (Oxford Optronix, Abingdon, United Kingdom).
2.9. Western blotting
Protein samples were analyzed by SDS-PAGE and Western blotting
with the following antibodies: anti-Myb (5E11, [40]), anti-β-actin (Sig￾ma-Aldrich, AC-15), anti-Gal4 (SantaCruz Biotechnology, RK5C1),
anti-p300 (Millipore, RW128), anti-GFP (clones 7.1 and 13.1, Roche
Diagnostics), anti-HA (BioLegend, 16B12) and anti-acetyl-Lysine (Cell￾Signaling, Ac-K-103).
Fig. 1. Oprozomib targets the transactivation domain of MYB. A. Schematic illustration of the HEK-MYB-Luc and HEK-Luc reporter cell lines and the candidate
MYB inhibitors identified by library screening. B. HEK-Luc and HEK-MYB-Luc cells were treated for 16 h with doxycycline and oprozomib at the indicated con￾centrations. Bars show the average luciferase activity of the cells relative to cells treated only with doxycycline. The bottom panels show the expression of MYB and
β-actin. C. HEK293T cells were transiently transfected with the MYB-responsive reporter pGL4-5xMRE(GG)-Myc and expression vectors for wt MYB, MYB-2KR and
MYB-CT3. Transfected cells were distributed in identical aliquots into microtiter plates and treated with the indicated concentrations of oprozomib. Cells were
harvested after 16 h and used for luciferase assays and western blotting to determine MYB expression. Bars show the average luciferase activity of the cells
normalized to untreated cells. Protein gels containing MYB, MYB-2KR, MYB-CT3 and β-actin are shown at the bottom. D. EMSA experiments with nuclear extracts
from HEK293T cells transfected with an expression vector for C-terminally truncated MYB (MYB-CT3) and cultivated for 16 h with the indicated oprozomib con￾centrations are shown on the left. Nuclear extract from un-transfected HEK293T cells was used as additional control. Binding assays were performed with a radi￾olabeled oligonucleotide containing a consensus MYB binding site. A Western blot showing MYB-CT3 expression in aliquots of the nuclear extracts is presented at the
top. In addition to using nuclear extracts from cells treated with oprozomib in vivo we performed EMSA experiments by adding oprozomib directly to the in vitro
binding reactions (last three lanes of the EMSA panel). MYB-specific protein-DNA-complexes are labeled by black dots. Densitometric analysis of band intensities of
the slower-migrating MYB-specific complex showed that it was reduced from 100% (without oprozomib) to 97% at 300 nM or to 94% at 5 μM oprozomib in the in
vivo or in vitro experiment, respectively. E. Luciferase reporter experiments of HEK293T cells transfected with the Gal4-dependent reporter pG5E4-38Luc and
expression vector for a Gal4-CT3 fusion protein. The cells were treated for 16 h without or with the indicated concentrations of oprozomib, followed by analysis of the
luciferase activity (top) and the expression of Gal4-CT3 and β-actin (bottom). Asterisks in panels B to E indicate statistical significance (ns: non-specific; *p < 0.05;
**p < 0.01; ***p < 0.001; Student’s t-test).
2.10. Statistical analysis
All experiments subjected to statistical analysis were performed at
least three times with independent replicates in each experiment. Data
were shown as mean ± standard deviation, which reflects the variation
within each group. Statistical differences between groups were calcu￾lated by the two-tailed Student’s t-test or by one-way ANOVA. Values of
P < 0.05 were considered as statistically significant.
3. Results
3.1. Screening of a compound library with a MYB reporter cell line
To identify novel MYB inhibitors we have used the previously
developed MYB reporter cell line HEK-MYB-Luc, which allows
doxycycline-inducible expression of an activated version of human MYB
(referred to as MYB-2KR) (Fig. 1A). This cell line also harbors a lucif￾erase reporter gene driven by a minimal promoter and several copies of a
high-affinity MYB binding site [33]. MYB-2 KR is a
sumoylation-deficient MYB mutant that has a higher transactivation
potential than wild type MYB [34,36]. Using these cells we screened a
library of approximately 4000 biology-annotated compounds, including
many FDA-approved drugs, for novel MYB-inhibitory compounds. Initial
screening was performed in 384-well microtiter plates at 10 μM com￾pound concentration using monensin, a previously identified
MYB-inhibitory agent [32], as a positive control to confirm the repro￾ducibility of the assay. Primary screening identified several candidate
inhibitory compounds, which were retested in triplicates at 10 μM
concentration, using HEK-MYB-Luc and HEK-Luc cells. HEK-Luc cells
express firefly luciferase from a constitutive promoter, allowing exclu￾sion of toxic compounds and unspecific luciferase inhibitors. The
candidate inhibitors were finally subjected to IC50 validation and cell
numbers were analyzed for evaluation of assay interference. The pro￾teasome inhibitors oprozomib, carfilzomib and ixazomib showed the
highest MYB-inhibitory activity and were selected for further studies
(Fig. 1A). We confirmed their MYB inhibitory potential by testing them
again in luciferase assays, using HEK-MYB-Luc and HEK-Luc cells.
Analysis of total cell extracts of HEK-MYB-Luc cells for MYB and β-actin
expression showed that the proteasome inhibitors strongly increased
MYB expression at higher concentrations (Fig. 1B and Supplementary
Fig. 1). For further analysis, most subsequent experiments focused on
oprozomib as one of the highly active proteasome inhibitors.
3.2. Oprozomib affects the function of the MYB transactivation domain
Since the HEK-MYB-Luc cells express a mutated version of human
MYB we next investigated the effect of oprozomib on wild-type human
MYB. We transfected expression vectors for MYB-2KR and wild-type
MYB together with the MYB-dependent luciferase reporter construct
transiently into HEK293T cells and determined the effect of different
concentrations of the compounds (Fig. 1C). We also assessed the ability
of the compounds to inhibit the activity of the C-terminally truncated
mutant MYB-CT3. Because the sequences responsible for ubiquitin￾dependent degradation of MYB reside in its C-terminal part [41], the
effect of oprozomib on the truncated MYB would tell us if the inhibition
of MYB activity was due to its increased expression, for example by
titrating out a limiting co-factor. Oprozomib inhibited wild-type MYB
only slightly less efficiently than MYB-2KR. As expected, the expression
of MYB-CT3 was not increased by oprozomib, however, its activity was
strongly inhibited, excluding that the inhibition of MYB activity was due
to the increased amount of MYB (Fig. 1C). To investigate which func￾tional domains of MYB mediated the inhibitory effect of oprozomib, we
focused on the transactivation and DNA-binding domains. Nuclear ex￾tracts prepared from cells expressing MYB-CT3 and treated for 16 h with
relevant oprozomib concentrations were used to perform electropho￾retic mobility shift assays (EMSA) to investigate MYB DNA-binding
activity. The EMSA experiments revealed several complexes of the
MYB-binding site-containing oligonucleotide upon incubation with nu￾clear extract, two of which (labeled by black dots in Fig. 1D) were
present only when extracts from cells expressing MYB-CT3 were used.
This indicated that these are complexes of MYB-CT3 with the binding
site oligonucleotide, and that the slower and faster migrating complex
presumably is derived from full-length or partially degraded MYB-CT3.
The EMSA experiments showed only marginal inhibition of the
DNA-binding activity at oprozomib concentrations that strongly reduced
the activity of MYB-CT3. We also performed EMSA experiments in which
nuclear extracts from MYB-CT3 expressing cells were treated with
significantly higher optozomib concentrations in vitro. However, the
compound also failed to significantly interfere with MYB DNA-binding
activity under these conditions. In additional experiments, we
replaced the DNA-binding domain of MYB-CT3 by the yeast GAL4
DNA-binding domain and investigated whether the activity of the fusion
protein was still inhibited by oprozomib. As shown in Fig. 1E, the ac￾tivity of the GAL4-MYB fusion protein containing the MYB trans￾activation domain was inhibited by oprozomib in a
concentration-dependent manner. Overall, these experiments strongly
suggest that oprozomib inhibits MYB activity via the MYB trans￾activation domain.
3.3. Oprozomib disturbs the stimulation of MYB activity by the co￾activator p300
The transactivation potential of MYB critically depends on the
interaction of the MYB transactivation domain with the co-activator
p300/CBP [19]. We performed luciferase assays of cells transfected
with MYB-2KR alone or together with p300 to investigate if oprozomib
interferes with the cooperation of MYB and p300 (Fig. 2A and B). The
overall MYB activity was substantially increased by co-expression of
p300, consistent with the cooperation of both proteins. Importantly,
oprozomib suppressed this increase in a concentration-dependent
manner. When normalized to the luciferase activity of the cells in the
absence of oprozomib, the concentration-dependent inhibition by
oprozomib was virtually identical in the presence or absence of ectopic
p300, supporting the notion that oprozomib disturbs the function of
p300 as a co-activator of MYB. Similar observations were made for
carfilzomib and ixazomib (Supplementary Fig. 2). Single amino acid
replacements of methionine-303 by valine (M303V) or leucine-302 by
alanine (L302A) are known to weaken the binding of p300 and decrease
the transactivation potential of MYB [21,42]. As expected, when intro￾ducing these mutations into MYB-CT3 they both reduced MYB activity
(Fig. 2C). Notably, MYB-CT3(L302A) was virtually inactive indicating
that p300 or its paralog CREB-binding protein (CBP) is the major
co-activator responsible for the transcriptional activity of MYB. We
compared the inhibitory effects of oprozomib on wild-type MYB-CT3
and the M303V mutant, thereby mimicking a decrease of the endoge￾nous p300. To facilitate this comparison, the activity of both versions of
MYB-CT3 was compared side-by-side by normalizing the activity in the
absence of inhibitor to 100% in both cases (Fig. 2D). This showed that
the extent of inhibition at each concentration of oprozomib was iden￾tical for wild-type and mutant MYB, again supporting the notion that
oprozomib interferes with the stimulation of MYB activity by p300.
The amino acid sequence around the p300-binding region in the
transactivation domain of the MYB family member MYBL1 (A-MYB) is
very similar to that of MYB. Furthermore, MYBL1 has also been shown to
cooperate with p300 [43]. As expected, oprozomib also inhibited the
activity of MYBL1 in a concentration-dependent manner (Fig. 2E and F).
3.4. Oprozomib targets the transactivation potential of the C-terminal
p300 domain
As a first step to address how oprozomib inhibits the cooperation of
MYB and p300 we asked if the expression of endogenous p300 was
M.V. Yusenko et al.
Cancer Letters 520 (2021) 132–142
136
affected by oprozomib. Fig. 3A shows that concentrations of oprozomib
that strongly suppress MYB activity did not decrease but rather slightly
increased the level of endogenous p300 expression. We then asked if
oprozomib interferes with the recruitment of p300 to MYB. To address
this, we employed a co-precipitation approach in which extracts from
cells expressing p300 and a GFP fusion protein containing the MYB
transactivation domain were subjected to GFP-trap to analyze the in vivo
binding of p300 to the GFP-fused MYB transactivation domain. An initial
GFP-trap experiment confirmed that the transactivation domain was
able to recruit p300 (Fig. 3B). We then performed a GFP-trap experiment
in the absence or presence of oprozomib, using a concentration that
results in almost complete inhibition of MYB activity (as seen in Fig. 2A).
Although the amount of p300 in the GFP-trap experiment was slightly
lower in the presence of oprozomib than in its absence, densitometric
scanning of the input and trap bands showed that the interaction be￾tween MYB and p300 was only slightly reduced by the compound
(Fig. 3C). Thus, the virtually complete inhibition of MYB activity by
oprozomib cannot be explained by a disruption of the MYB-p300
interaction. We therefore turned to the possibility that the acetylation
of MYB by the HAT activity of p300 [44] was inhibited by oprozomib.
However, oprozomib had no significant effect on the acetylation level of
MYB (Fig. 3D). We also examined the acetylation of the C-terminally
truncated MYB-CT3 by p300, which lacks previously identified acety￾lation sites [44] and was therefore not acetylated by p300 (Fig. 3E). The
observation that the activities of MYB-CT3 and wild-type MYB are
inhibited by oprozomib in similar manner (see Fig. 1C) argues against a
model in which the inhibitory effect of oprozomib is due to changes of
the level of MYB acetylation. Taken together, the inhibitory activity of
oprozomib appeared not to be due to decreased MYB-p300 interaction
or decreased acetylation of MYB.
The C-terminal domain of p300 has previously been shown to
interact with certain co-activators and to stimulate transcription when
linked to a DNA-binding domain [45,46]. To examine the trans￾activation potential of the C-terminal part of p300 we performed re￾porter experiments with an expression vector for a Gal4-p300
(1818–2414) fusion protein and a GAL4-dependent luciferase reporter.
As shown in Fig. 3G, oprozomib strongly inhibited the transactivation
potential of the C-terminal part of p300 in a concentration-dependent
manner. Similar observations were also made for carfilzomib and ixa￾zomib (Supplementary Fig. 3). These data raise the possibility that
proteasome inhibitors suppress MYB activity by interfering with the
transactivation function of the C-terminal domain of p300.
Since p300 cooperates with various transcription factors we
wondered whether oprozomib inhibits their activity as well. As a first
Fig. 2. Oprozomib disturbs the stimulation of MYB activity by co-activator p300. A. HEK293T cells were transiently transfected with the MYB-dependent
reporter gene pGL4-5xMRE(GG)-Myc and expression vectors for MYB-2KR and p300, as shown at the top. Transfected cells were treated with the indicated opro￾zomib concentrations. Cells were harvested after 16 h and used for luciferase assays. The top panels show the absolute luciferase values in arbitrary numbers and the
bottom panels show the luciferase activity normalized to that of untreated cells. B. Western blot analysis of p300 in HEK293T cells transfected with p300 expression
vector and treated for 16 h with the indicated concentrations of oprozomib. C. Luciferase reporter assays of HEK293T cells transfected with expression vectors for
MYB-CT3 (black bars) and MYB-CT3(M303V) or MYB-CT3(L302A) (white bars). D. HEK293T cells were transfected with reporter gene pGL4-5xMRE(GG)-Myc and
expression vectors for MYB-CT3 or MYB-CT3(M303V) and treated without or with the indicated concentrations of oprozomib for 16 h before harvesting and
measuring luciferase activities. The difference in activity between MYB-CT3 and the M303V mutant was compensated by expressing the luciferase activities as
percent of the activity of the untreated cells. E. HEK293T cells were transfected with reporter gene pGL4-5xMRE(GG)-Myc and expression vectors for human MYBL1
(A-MYB) and treated without or with the indicated concentrations of oprozomib for 16 h before harvesting and measuring luciferase activities. F. Protein analysis of
A-MYB expressing cells described in panel E. Asterisks in panels A and C to E indicate statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001; Student’s t-test).
M.V. Yusenko et al.
step to address this issue we examined its effect on the activity of C/
EBPβ, a transcription factor known to cooperate with MYB in myeloid
cells [37–39]. We found that C/EBPβ was also inhibited by oprozomib
(Supplementary Fig. 4), but that its effect at low nanomolar concen￾trations was less strong than in case of MYB (compare Fig. 2A and
Supplementary Fig. 4).
3.5. Oprozomib down-regulates MYB target genes and induces expression
of the myeloid differentiation marker CD11b and cell death in AML cells
To characterize the MYB inhibitory potential of oprozomib in a
biologically relevant setting we studied its effects on human AML cell
lines. We first examined the effect of oprozomib on the viability of MYB￾expressing AML cell lines and several non-hematopoietic cell lines. This
showed that the AML cell lines were more sensitive to the compound
than the non-hematopooietic cells, consistent with its MYB inhibitory
activity (Fig. 4A). Treatment of THP1 cells with oprozomib resulted in
Fig. 3. Oprozomib interferes with the activity of the C-terminal domain of p300. A. Expression of endogenous p300 in HEK293T cells treated for 16 h with or
without oprozomib. B. HEK293T cells were transfected with expression vectors for HA-tagged p300, GFP or GFP-MYB(205–442). The cells were harvested after 16 h
and total cell extract was subjected to GFP-trap. Aliquots of the total cell extracts and samples of the bound proteins were then analyzed by Western blotting for p300
and GFP-MYB(205–442). C. HEK293T cells transfected with expression vectors for HA-tagged p300 and GFP-MYB(205–442) and treated with or without 0.3 μM
oprozomib. The cells were then analyzed by GFP-trap as in panel B. For convenience, exposure times of TCE and trap samples were adjusted such that the p300 bands
in input and bound p300 in the absence of oprozomib were of similar intensity. The numbers below the bands indicate the relative band intensities (determined by
ImageJ) between untreated (set at 100%) and oprozomib-treated samples. Based on these numbers, the efficiency of co-precipitation was reduced by approximately
14% in the presence of 0.3 μM oprozomib (setting the reduced input in the presence of oprozomib as 100%). D. HEK293T cells were transfected with expression
vectors for p300 and MYB and treated for 16 h with the indicated concentrations of oprozomib. In the left panel aliquots of total cell extract were then analyzed by
Western blotting for the expression of p300, MYB and acetylated MYB. In the right panel, aliquots of total extracts of cells transfected with expression vectors for MYB
and p300 and treated with oprozomib were analyzed identically. Non-transfected cells are shown as additional control. E. HEK293T cells were transfected with
expression vectors for p300, wild-type MYB and MYB-CT3, as indicated. Total cell extracts were analyzed after 16 h with antibodies against MYB and acetylated
lysine. F. HEK293T cells were transfected with the Gal4-inducible reporter gene pG5E4-38Luc and expression vector for a Gal4-p300(1752–2514) fusion protein
shown schematically at the top. The cells were treated for 16 h without or with the indicated oprozomib concentrations, followed by analysis of the luciferase activity
(bottom left) and the expression of Gal4-fusion protein (bottom right). Asterisks in panel E indicate statistical significance (**p < 0.01; ***p < 0.001; Student’s
t-test).
down-regulation of the direct MYB target genes MYC, GFI1, and KIT
already after 2 h of treatment (Fig. 4B). This supports the notion that the
down-regulation of their expression by oprozomib is a directly effect, in
accordance with the MYB-inhibitory activity of the compound, rather
than an indirect consequence of the differentiation of the cells. To
support this further, we have also recapitulated the inhibitory effect of
oprozomib on GFI1 expression in a reporter experiment by employing a
luciferase plasmid that contains the GFI1 promoter and encompasses a
previously identified MYB binding site [47]. Fig. 4C shows that the ac￾tivity of the reporter was stimulated by MYB, particularly in the pres￾ence of p300, and that the promoter activity in the presence of MYB and
p300 was decreased by oprozomib in a concentration-dependent
manner.
In AML cells, MYB is known to control a transcriptional program that
maintains the viability of the cells and prevents differentiation. Inhibi￾tion of MYB is therefore expected to trigger differentiation and/or cell
death. Treatment of HL60 cells with oprozomib showed that the myeloid
differentiation marker CD11b was up-regulated by oprozomib in a
concentration dependent manner (Fig. 4D). We also examined this
ability of oprozomib on HL60 cells ectopically expressing a C-terminally
truncated, activated version of MYB [48]. As seen in Fig. 4D, the pres￾ence of activated MYB diminished CD11b expression, indicating that the
ability of oprozomib to trigger CD11b expression is mediated to a sig￾nificant extent by the inhibition of MYB activity. Oprozomib also
induced cell death in HL60 cells in a concentration-dependent manner,
which again was counteracted by ectopic expression of C-terminally
truncated MYB (Fig. 4D, lower panel). Finally, we examined HL60 cells
for morphological signs of differentiation induced by oprozomib. Fig. 4E
shows that the oprozomib-treated cells were slightly enlarged with an
increased cytoplasmic-to-nuclear ratio.
Fig. 4. Oprozomib inhibits expression of MYB target genes, induces myeloid differentiation marker CD11b and cell death in AML cells. A. Inhibition of cell
viability by oprozomib. Non-hematopoietic cell lines (HEKT, U2OS, HepG2, HeLa) and MYB-expressing AML cell lines (U937, HL60, THP1, NB4) were treated for 24
h with the indicated concentrations of oprozomib and analyzed by a MTT assay. The figure shows percent viable cells (with standard deviation) relative to untreated
cells. B. Down-regulation of MYC, GFI1 and KIT mRNA expression in THP1 cells treated for 2 h with 100 nM oprozomib. Gene expression was determined by RT-PCR
and normalized to β-actin expression. C. Luciferase reporter assay of the human GFI1 promoter. Columns on the left: Hek293T cells were transfected with the
luciferase plasmid pGL4-huGFI1(KpnI/ApaI) and expression vectors for MYB and p300, as indicated. Luciferase activity was determined after 16 h. Columns on the
right: HEK293T cells were transfected with the GFI1 luciferase reporter plasmid and expression vectors for MYB and p300. Cells were treated with the indicated
concentrations of oprozomib for 16 h before measuring the luciferase activity. D. HL60 cells infected with a control lentivirus or a lentivirus encoding C-terminally
truncated MYB (HL60-CT3) were treated for 48 h with the indicated concentrations of oprozomib. The cells were then analyzed by flow cytometry for CD11b
expression (top left panel) or for annexin-V and propidium iodide staining (bottom panel), and by Western blotting for MYB and β-actin expression (right panels). E.
May-Grünwald Giemsa staining of HL60 cells cultivated for 2 days without or with 30 nM oprozomib. Asterisks in panels B to D indicate statistical significance (ns:
non-specific; *p < 0.05; **p < 0.01; ***p < 0.001; Student’s t-test).
M.V. Yusenko et al.
3.6. Oprozomib suppresses proliferation of ACC cells
As a first step to investigate the effects of proteasome inhibition on
ACC we used patient-derived ACC cells from two cases expressing MYB￾NFIB fusion proteins to examine the impact of oprozomib on their pro￾liferation. Fig. 5A shows that the proliferation of ACC cells was strongly
inhibited at nanomolar concentrations of oprozomib whereas non￾malignant pleomorphic adenoma (PA) cells used as control were
significantly less sensitive. The concentration of oprozomib at which
50% inhibition occurs is estimated to be approximately 50 and 20 nM for
ACC1 and ACC2 cells, respectively, and 400 nM for PA cells. This
showed that the same degree of inhibition is observed only at an 8- to 20-
fold higher compound concentration in the control cells. Oprozomib also
induced apoptosis in both ACC cases more efficiently than in PA cells,
with ACC2 cells again being particularly sensitive (Fig. 5B). Sphere￾forming assays with ACC2 cells to assess the effect of oprozomib on
their self-renewing capacity showed a strong decrease in the number as
well as the size and density of spheres in the presence of oprozomib,
supporting the result of the viability assay (Fig. 5C–F). Taken together
with the inhibitory effects observed on AML cells, these results show for
the first time that cells derived from two different maligancies that share
a high dependence on MYB are highly sensitive to nanomolar concen￾trations of oprozomib. Overall, this makes future studies of the thera￾peutic potential of proteasome inhibitors for MYB-driven tumors highly
interesting.
4. Discussion
Recent insight into the role of MYB in human malignancies has
highlighted MYB as a potential drug target for therapy of AML and ACC.
Efforts to target MYB by low molecular weight compounds have proven
successful and have provided initial evidence for the therapeutic po￾tential of MYB inhibition [4,26–30]. To expand the repertoire of
MYB-inhibitory compounds we have screened a library of FDA-approved
drugs with a MYB reporter cell line, which allowed us to identify pro￾teasome inhibitors as novel agents that suppress MYB activity at nano￾molar concentrations.
The ubiquitin-proteasome system is one of the major proteolytic
systems in the cell whose dysfunction has been implicated in many
diseases, which has made the proteasome an interesting target for
Fig. 5. Effects of oprozomib on patient-derived
ACC cells. A. Cell proliferation assay of ACC cells
and pleomorphic adenoma (PA ctrl) cells treated
with oprozomib for 72 h. B. Induction of apoptosis
by oprozomib in ACC cells and PA control cells.
Cells were treated for 24 h. Asterisks indicate sta￾tistical significant differences between ACC and
control cells for each concentration (***p < 0.001;
****p < 0.0001; one-way ANOVA). C. Sphere￾forming assay of ACC2 cells after 10 days in the
presence of 100 nM oprozomib. D-F. Quantification
of the number, the average diameter, and the opti￾cal density of spheres formed by ACC2 cells in the
absence or presence of 100 nM oprozomib. Aster￾isks in panels E and F indicate statistical signifi￾cance (***p < 0.001; ****p < 0.0001; Student’s t￾test).
M.V. Yusenko et al.
therapeutic approaches. Proteasome inhibitors have already been
studied as therapeutic agents and have become mainstays for the
treatment of multiple myeloma and mantle cell lymphoma [49,50].
Interestingly, they have also yielded encouraging results in clinical trials
of AML, especially in combination with chemotherapeutic drugs [51,
52]. Proteasome inhibitor treatment of AML cells was shown to inhibit
NF-kB, a transcription factor that is constitutively active in AML stem
cells due to an autocrine TNFα-mediated feedback loop, and promotes
cell survival and proliferation [53,54]. Another effect of proteasome
inhibition in AML cells is the autophagy-dependent degradation of the
mutant FLT3 receptor (FLT3-ITD), which is found in a significant per￾centage of AML-patients [55].
Our work highlights the suppression of MYB activity as a novel
consequence of proteasome inhibition and suggests that proteasome
inhibition interferes with the stimulation of MYB activity by co-activator
p300. Previous work has uncovered multiple links between proteins
involved in transcriptional activation and components of the ubiquitin￾proteasome system [56,57]. This has inspired a model of activator
regulation in which transcription factors, as usually short-lived proteins,
are capably of stimulating transcription only briefly and are marked for
degradation by the transcriptional activation process itself. In this
model, proteasomal degradation is required for clearing “used” activator
proteins that are trapped in an inactive state, thereby allowing “pristine”
activator molecules to bind to promoters and stimulate further rounds of
transcription [56,57]. Although this idea could explain why blocking
proteasomal degradation inhibits the activity and simultaneously in￾creases the amount of a transcription factor, in case of MYB this model is
difficult to reconcile with the observation that MYB-CT3, which lacks
the sequences responsible for ubiquitin-dependent degradation [41], is
inhibited by oprozomib like full-length MYB (Fig. 1C). Because the
suppression of MYB activity by different proteasome inhibitors (Fig. 1
and Supplementary Fig. 1) roughly paralleled the increased MYB
expression, we initially considered the possibility that the decrease of
MYB activity by proteasome inhibitors was due to the titration of a
limiting cofactor, such as p300, due to the increased amount of MYB or
other short-lived cellular proteins recruiting p300. Although we cannot
completely exclude such a mechanism several observations argue
against it. The fact that oprozomib also inhibits the activity of the
C-terminally truncated MYB makes it unlikely that the inhibition is due
to titration of a limiting co-factor due to the excess of MYB itself
(Fig. 1C). Furthermore, the MYB mutant M303V, which binds p300 less
strongly than wild-type MYB, was inhibited by oprozomib in a similar
concentration-dependent manner as wild-type MYB. If the effect of the
proteasome inhibitor was to increase expression of other cellular pro￾teins recruiting p300, one might have expected to see a difference in the
concentration-dependent inhibition by oprozomib between wild-type
and mutant MYB (Fig. 2D). Furthermore, overexpression of p300 did
not change the concentration-dependent inhibition by oprozomib
(Fig. 2A). If oprozomib simply increased the expression level of cellular
proteins competing for a limited amount of p300, one would expect to
see a less strong inhibitory effect when overexpressing p300.
How oprozomib disturbs the ability of p300 to stimulate MYB ac￾tivity is still not known. Oprozomib had only a weak effect on the
interaction of MYB and p300. Furthermore, our data argue against the
notion that the inhibition of MYB activity by oprozomib is due to
decreased acetylation of MYB (Fig. 3C–F), raising the possibility that
oprozomib disturbs another function or molecular interaction of p300.
As a first clue supporting this possibility, we observed that oprozomib
interferes with the transactivation potential of the C-terminal part of
p300, which was demonstrated by fusing it to a DNA-binding domain
(Fig. 3E). Although the mechanism by which oprozomib inhibits the
activity of the C-terminal domain of p300 remains to be explored, these
observations are of interest because current approaches to develop
pharmacological inhibitors of p300 mainly focus on the HAT- and bro￾modomains of p300. Previous work has identified phosphorylation of
multiple sites in the C-terminal domain of p300 that are induced by
interaction with specific transcription factors and may modulate the
function of this domain [58,59]. Protein structure predictions suggest
the C-terminal part of p300 to be intrinsically disordered, raising the
possibility that its function is related to the formation of biomolecular
condensates at so-called super-enhancers [60,61]. In this regard, it is
interesting to note that the oncogenic activity of MYB has repeatedly
been linked to the formation of super-enhancers at critical target genes
[12,13,16]. It will be interesting to investigate in future work if and how
oprozomib interferes with any of these aspects of p300.
The notion that oprozomib inhibits MYB via p300 also raises the
question whether other transcription factors are also inhibited by p300-
dependent mechanisms. As a first step to address this question we have
found that C/EBPβ was also inhibited, however, differences in the
concentration-dependence of the inhibitory effect of oprozomib be￾tween MYB and C/EBPβ suggest that different mechanisms may be
involved. We recently showed that C/EBPβ plays a pro-oncogenic role as
a cooperation partner of MYB in AML cells [62], suggesting that the
simultaneous inhibition of both factors may actually be beneficial for the
effects caused by oprozomib in AML cells. However, on a broader scale it
remains to be investigated in future work if and how other transcription
factors interacting with p300 are inhibited by oprozomib.
To explore the biological effects of oprozomib we have employed
cultured AML and ACC cells, representing two different human malig￾nancies driven by deregulated MYB expression. In THP1 cells, oprozo￾mib down-regulated several MYB target genes within 2 h, confirming the
MYB-inhibitory activity of oprozomib in a physiological setting. The fast
time course of the down-regulation makes it unlikely that this is an in￾direct effect caused by the differentiation of the cells. Furthermore, the
use of a GFI1 promoter construct allowed us to recapitulate the inhibi￾tory effect of oprozomib in a reporter experiment, substantiating its
inhibitory activity. Prolonged treatment of HL60 cells resulted in in￾duction of the expression of the differentiation-associated CD11b gene
and in cell death, as expected if MYB is inhibited. Notably, these effects
were significantly suppressed by ectopic expression of C-terminally
truncated MYB, indicating that they were mediated to a significant part
by decreased MYB activity.
ACC is a rare cancer with a high potential of recurrence and metas￾tasis and with no treatment options for patients with advanced disease.
So far, no effective systemic or targeted therapies have been established
for ACC. In light of these limitations it is of considerable interest to note
that oprozomib disrupts MYB activity and induces strong inhibitory ef￾fects on the proliferation of patient-derived ACC cells. These effects were
induced at significantly lower compound concentrations in ACC cells
than in non-malignant pleomorphic adenoma control cells. They were
also supported by sphere-forming assays, confirming significant inhibi￾tion of the self-renewal capacity of ACC stem cells by oprozomib. In
summary, our work reveals a novel link between proteasome inhibition
and MYB activity by showing that proteasome inhibitors are particularly
potent MYB-inhibitory agents that act in a p300-dependent manner and
inhibit AML and ACC cell proliferation. Thus, our work provides novel
insight into the complex effects elicited by proteasome inhibition.
Further studies of the molecular and biological effects of proteasome
inhibition and their potential therapeutic relevance for MYB-driven
malignancies will therefore be of high interest.
Availability of data and materials
All data are presented in this article and the supplementary data file.
Funding
This work was supported by grants from the Deutsche Krebshilfe, the
Else Kroner-Fresenius ¨ Stiftung and the Deutsche Jose Carreras
Leuk¨
amie-Stiftung e. V, the Swedish Cancer Society, the Swedish
Childhood Cancer Fund, and the Sjoberg ¨ Foundation.
M.V. Yusenko et al.
Cancer Letters 520 (2021) 132–142
Author contributions
M.V.Y. and A.B. designed and performed experiments and analyzed
data; J.P.v.K. and S.R. designed and performed screening experiments
and analyzed data, M.K.A. and G.S. provided ACC cells, designed and
performed experiments, analyzed data and wrote the manuscript, K.-H.
K. conceived the study, designed and performed experiments, analyzed
data and wrote the manuscript. All authors approved the final version of
the manuscript.
Declaration of competing interest
The authors declare that they do not have any conflict of interest to
disclose.
Acknowledgements
We thank B. Berkenfeld for expert technical assistance.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.canlet.2021.07.010.
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