Relationship between inhibition profile of β1, β2 and β5 proteasome subunits and cytotoxic activity of
proteasome inhibitors in Multiple Myeloma
1
1
1
1
2
2
2
1
Andrej Besse* , Marianne Kraus , Jurgen Bader , Lenka Besse , Bo-Tao Xin , Gerjan de Bruin , Herman S. Overkleeft , Christoph Driessen
1
Hematology/Oncology, Kantonsspital St Gallen, St. Gallen, Switzerland
2
Gorlaeus Laboratories, Leiden Institute of Chemistry and Netherlands Proteomics Centre, Leiden, Netherlands
BACKGROUND
β1i
β5i
β1/1i
β2/2i
β5/5i
AMO-1 aBtz
Ø
β1i
β5i
β1/1i
β2/2i
β5/5i
AMO-1 aCfz
Ø
β1i
β5i
β1/1i
β2/2i
β5/5i
Ø
β1i
β5i
β1/1i
β2/2i
100.0
103.5
106.9
114.7
100.5
111.6
115.8
77.2
74.6
40.8
36.8
12.7
12.7
14.0
12.7
12.8
Ø
β1i
β5i
β1/1i
β2/2i
100.0
95.9
100.3
95.9
93.5
98.5
93.2
87.4
94.5
89.9
87.3
97.6
96.3
96.6
88.7
17.0
Ø
β1i
β5i
β1/1i
β2/2i
100.0
96.2
100.7
94.5
91.2
92.5
90.6
93.2
95.8
95.3
92.4
95.0
95.6
93.1
92.2
89.8
β2/2i+β1/1i+β5i
β2/2i+β1/1i+β5/5i
21.9
12.5
β2/2i+β1/1i+β5i
β2/2i+β1/1i+β5/5i
88.9
18.4
β2/2i+β1/1i+β5i
β2/2i+β1/1i+β5/5i
94.7
90.7
ARH-77
Ø
β1i
β5i
β1/1i
β2/2i
β5/5i
ARH-77 aBtz
Ø
β1i
β5i
β1/1i
β2/2i
β5/5i
Ø
β1i
β5i
β1/1i
β2/2i
β5/5i
Ø
β1i
β5i
β1/1i
β2/2i
100.0
97.5
106.3
104.7
94.5
104.5
97.5
92.8
102.7
95.8
78.3
21.7
21.2
21.4
19.5
21.2
Ø
β1i
β5i
β1/1i
β2/2i
100.0
99.9
102.1
115.2
96.4
109.0
103.7
98.3
102.7
105.0
86.9
94.2
104.9
95.7
93.3
30.0
ARH-77 aCfz
Ø
β1i
β5i
β1/1i
β2/2i
100.0
104.1
105.8
109.8
102.8
111.1
108.7
96.5
103.3
101.8
93.2
106.3
112.7
106.1
80.6
25.5
β2/2i+β1/1i+β5i
β2/2i+β1/1i+β5/5i
80.4
19.3
β2/2i+β1/1i+β5i
β2/2i+β1/1i+β5/5i
117.5
32.4
RPMI8226
Ø
β1i
β5i
β1/1i
β2/2i
β5/5i
Ø
β1i
β5i
β1/1i
β2/2i
100.0
98.5
91.7
105.3
95.7
99.7
94.5
67.5
66.8
51.9
37.9
17.5
19.0
19.7
16.6
21.9
β2/2i+β1/1i+β5i
β2/2i+β1/1i+β5/5i
32.2
16.1
2
2i
5i
5
1
1i
5i
5
1
1i
5i
5
1
1i
5i
5
1
1i
5i
5
1
1i
400
800 [nM] F i g . 3 .
Inhibition profile of
proteasome subunits by currently
clinically available or tested PI in
intact cells.
All PI in clinical use or development
target primarily β5/5i, but in addition
co-target also β1/1i, β2/2i subunits at
higher concentrations to variable
degrees. Only Cfz, which is partly able
to overcome Btz-resistance in
patients, shows in addition inhibition
of β2/2i subunits. Thus, we developed
Xin 550/551 as new PI co-targeting
β5/5i and β2/2i subunits.
1600 3200 [nM]
Viability [%]
200
Xin551
2
2i
100
Xin550
2
2i
50
Oprozomib
2
2i
25
Ixazomib
2
2i
12.5
Carfilzomib overcomes Btz resistance, while Btz overcomes
Cfz-resistance in vitro.
Co-inhibition of β2/2i proteasome activity increases the
cytotoxicity of all clinical stage proteasome inhibitors and
overcomes BTZ resistance
The β2/2i/β5/5i targeted novel PI Xin550/551 induce toxicity
also in Cfz resistant cells
REFERENCES
XIN550
5i
5
1
1i
Viability [%]
120%
100%
80%
60%
40%
20%
0%
Fig.3. Clinical stage PI drugs, except Cfz, inhibit mostly β5/5i
without significant co-inhibition of β1/1i and/or β2/2i. Cfz is the
only PI co-inhibiting β2/2i. Xin550/551 provide potent coinhibition of β5/5i, β2/2i subunits at high concentrations.
CONCLUSIONS
XIN551
2
2i
119.2
21.3
β-subunits inhibition profile of different PI
its
su
bu
n
β2
2i
0
β2/2i+β1/1i+β5i
β2/2i+β1/1i+β5/5i
Fig.2. β5 subunit inhibition is sufficient to induce cytotoxicity
in Btz-sensitive MM cells. Co-inhibition of β2/2i and β5 is
required for cytotoxicity in Btz-resistant MM cells. Inhibition
of β1/1i, with β5/5i or β2/2i, has no additional cytotoxic effect.
Fig.4. Co-inihibion of β2/2i potentiated the cytotoxicity of PI
againts Btz-adapted cells, but not Cfz-adapted cells. Xin550/
Xin551 are able to overcome the PI resistance of both, Btz- and
Cfz-adapted cells, when combined with a β2/2i inhibitor in vitro.
Carfilzomib
Ø
AMOaCfz
Delanzomib
Cfz-resitant
AMO-1
AMOaBtz
Bortezomib
AMO-1
Ixazomib
N
C
00
5
30
4
G
Lu
B
10
2
00
1
N
G
C
B
36
7
tr
ea
te
d
its
un
un
su
b
β-
5i
5
1
1i
Btz-resitant
Carfilzomib Delanzomib
We developed a set of subunit-selective PI as well as
fluorophore-labelled activity-based probes (ABP) capable
to visualize the individual activities of the β1/1i, β2/2i, β5/5isubunits of the constitutive- and immuno-proteasome in
intact cells. Equimolar concentrations of PI were used to
assess the inhibition profiles of the different drug
candidates. Btz/ Cfz-resistant MM cell lines were generated
by continuous drug exposure from AMO-1 cells (AMOaBtz/
AMOaCfz). Viability was estimated by MTS and plotted
against the inhibition of the individual β-subunits (alone or
with various combinations of proteasome inhibitors). In
addition, recently synthetized inhibitors with dual
specificity for β2/2i/ β5/5i (Xin550, Xin551) were used to
improve the efficacy of proteasome inhibition in PI
resistant cells.
2
2i
Oprozomib
sensitive
5i
5
1
1i
MATERIALS & METHODS
5i
5
1
1i
Impact of co-treatment with a β2/2i specific inhibitor on cytotoxicity
induced by established or candidate drug PI
FIg.4. AMO-1 sensitive cell line and its derivatives resistant to Btz or Cfz were
treated with increasing concentrations of different PI. Combination
treatment with Lu102, a β2/2i specific inhibitor, was able to decrease viability
in Btz adapted cells, but not of Cfz adapted cells. Treatment with Xin550/551
and Lu102 potentiated killing also in Cfz adapted cells.
Impact of selective inhibition of different β-subunits on viability of MM cells
Fig.2. Selective inhibition (> 80%) of the indicated subunits was reached and
combined for different proteasome inhibitors, subsequently cell viability was
measured.
Bortezomib
(I) To assess the impact of co-inhibition of proteasome
subunits activity (β1i, β1/1i, β2/2i, β5i, β5/5i) on cytotoxicity in Btz
sensitive and Btz-resistant MM cells; (II) to analyze the
subunit inhibition pattern of PI in clinical development in
relation to their cytotoxic activity.
2
2i
RPMI8226
OBJECTIVES
β-subunit selectivity of proteasome
inhibition
Fig.1. Inhibition profile of β-subunits of
proteasome and immunoproteasome in
two MM cell lines AMO-1 and RPMI8226
after treatment with subunit selective PI
(GB367 - β 1 i , NC001- β 1 / 1 i , Lu102- β 2 / 2 i ,
GB304-β5i, NC005-β5/5i) visualised by ABP
after labeling in intact cells.
AMO-1
The use of proteasome inhibitors (PI) with the first-in class
drug Bortezomib (Btz) has improved the outcome of
multiple myeloma (MM) patients. However, Btz resistance
frequently emerges in patients with advanced disease. The
second generation PI Carfilzomib (Cfz) and additional PI
(including Delanzomib (Dlz), Ixazomib (Ixa), Oprozomib
(Opro) ) are in advanced clinical development. All these PI
by design target the β5/5i of constitutive- and imunoproteasome (i) subunit which is the rate-limiting protease.
The proteasome in addition contains β1/1i and β2/2i protease
subunits that differ from β5/5i in their substrate specificity.
The degree of cytotoxicity of PI varies with the degree of coinhibition of proteasomal subunits β1/1i and/or β2/2i, in
addition to β5/5i. Btz-refractory MM cells upregulate activity
of β2/2i, the subunit not targeted by Btz. The “optimum”
pattern of proteasome inhibition to reach maximum
cytotoxicity in MM, either Btz-sensitive or -resistant is
unknown, as well as the individual patterns of proteasome
subunit inhibition (i.e. co-inhibition of β1/1i and/or β2/2i) of
the non-approved PI in MM.
SUMMARY
RESULTS
Concentration [nM]
AMO-1
AMOaBtz
AMOaCfz
IC50
Compound
[nM]
Compound +
Lu102 [nM]
Compound
[nM]
Compound +
Lu102 [nM]
Compound
[nM]
Compound +
Lu102 [nM]
Btz
3.1 (±0.2)
0.56 (±0.1)
298.7 (±8.7)
7.8 (±0.3)
27.9 (±0.5)
25.4 (±0.6)
Cfz
4.9 (±0.2)
0.75 (±0.1)
43.2 (±1.7)
3.5 (±0.1)
411.5 (±27.1)
283.6 (±10.6)
Dlz
5.9 (±0.3)
1.17 (±0.1)
>640
27.51 (±2.2)
143.2 (±4.6)
132.3 (±4.4)
Ixa
32.6 (±4.2)
10.362 (±0.9)
>640
82.4 (±4.9)
230.9 (±11.3)
194 (±6.9)
Opro
19.3 (±1..8)
3.4 (±0.2)
>640
70.5 (±4.2)
>640
>640
Xin550
21.4 (±1.2)
1.4 (±0.1)
310.1 (±12.1)
22.98 (±0.8)
247.5 (±35.3)
75.645 (±1.1)
Xin551
25.1 (±2.3)
1.38 (±0.1)
302 (±20.0)
27.4 (±1.1)
305.3 (±29.2)
122.15 (±7.3)
1. Ruckrich, T., Kraus, M., Gogel, J., et al. (2009) Characterization of the
ubiquitin-proteasome system in bortezomib-adapted cells. Leukemia.
23(6): 1098-105.
2. Britton, M., Lucas, M.M., Downey, S.L., et al. (2009) Selective
inhibitor of proteasome's caspase-like sites sensitizes cells to specific
inhibition of chymotrypsin-like sites. Chemistry & biology.
16(12):1278-89
3. Geurink, P.P., van der Linden, W.A., Mirabella, A.C., et al. (2013)
Incorporation of non-natural amino acids improves cell permeability
and potency of specific inhibitors of proteasome trypsin-like sites.
Journal of medicinal chemistry. 56(3):1262-75
4. Berkers, C.R., Verdoes, M., Lichtman, E., et al. (2005) Activity probe
for in vivo profiling of the specificity of proteasome inhibitor
bortezomib. Nature methods. 2(5): 357-62.
DISCLOSURES
The research reported in this poster was supported by Swiss
National Research Foundation SNF (grant 3100A_143924/ to C.D)
contact: [email protected]