TRIPLE COMBINATIONS OF THE HDAC INHIBITOR PANOBINOSTAT (NVP-LBH589) + DEXAMETHASONE WITH
EITHER LENALIDOMIDE OR BORTEZOMIB ARE HIGHLY EFFECTIVE IN A MULTIPLE MYELOMA MOUSE MODEL
Enrique M. Ocio1,2, David Vilanova2, Laura San Segundo2, Patricia Maiso2, Mercedes Garayoa2, Peter Atadja3, Atanasio Pandiella2, Jesús F. San Miguel1,2
1Hospital Universitario de Salamanca, Spain; 2Centro de Investigación del Cáncer. Universidad de Salamanca, Spain; 3Novartis Institute for Biomedical Research, Cambridge, MA
INTRODUCTION
MATERIAL & METHODS
Cancer therapy is more and more based on the concept of combining agents with different mechanisms of action in order to overcome
The in vitro activity of Panobinostat and its combinations was tested in the MM1S cell line by means of MTT and Annexin V staining by
and avoid the development of drug resistance. This is the rationale for most of the treatments currently used in hemato-oncology.
flow cytometry.
Multiple Myeloma is an incurable malignancy with a yearly incidence of 1 case per 10.000 inhabitants. Although important progress has
The in vivo efficacy was analyzed in a murine model of human subcutaneous plasmocytoma xenografted in NOD-SCID mice.
been achieved in recent years in the treatment and outcome of these patients, most of them still relapse after achieving response or are
120 mice were subcutaneously injected into the right flank with 3x106 MM1S cells. When tumors became palpable, mice were
refractory to the drugs used. This prompts the development of novel compounds that, in combination with the currently used agents,
randomized into 12 treatment groups including vehicle, drugs as single agents and double and triple combinations. Drugs were given ip,
could improve the response rates and prolong survival.
5 days/week x 7 weeks. Doses were: Panobinostat (P): 10 mg/Kg x 3 weeks and 5 mg/Kg afterwards; Dexamethasone (D): 1 mg/Kg;
Panobinostat (NVP-LBH589) is a novel deacetylase (DAC) inhibitor being evaluated in clinical trials in hematological and solid
Bortezomib (B): 0.1 mg/Kg; and Lenalidomide (L): 15 mg/Kg.
malignancies. We and others have previously demonstrated the potent in vitro antimyeloma activity of this agent.
Tumor volumes, clinical features and weight were monitored three times a week. Mice were sacrificed when their tumors reached 2
In the present study, the in vitro and in vivo antimyeloma effect of double and triple combinations of Panobinostat with conventional
cm and survival was assessed since the beginning of treatment. In selected tumors immunohistochemistry studies were performed.
antimyeloma agents has been explored.
IN VITRO RESULTS
Panobinostat as a single agent has important in vitro antimyeloma activity
The addition of Panobinostat to other antimyeloma agents, such as Dexamethasone, Lenalidomide or Bortezomib potentiates the in vitro activity of these
in MM cell lines sensitive and resistant to conventional treatments.
compounds. Triple combinations of Panobinostat with Dexamethasone and either Bortezomib or Lenalidomide are highly synergistic in an in vitro setting.
120
120
120
120
120
MM1S
)
)
100
100
MM1R
100
100
100
ls
ls
rol)
U266
control
80
control
80
cel
80
cel
80
80
U266LR7
cont
(%
(%
(+)
(+)
60
60
V
V
(%
U266DOX4
60
60
60
n
n
ake
40
40
40
40
40
uptake
uptake
nexi
nexi
n
n
upt
TT
20
TT
20
A
A
20
M
M
20
20
TTM
0
0
0
0
0
-
0
0.1
1
10
100
+
- +
- +
- + Panobinostat
- +
-
Panobinostat
+
- +
- +
- +
- +
- +
- + Panobinostat
- +
-
Panobinostat
+
- +
- +
Dexa
Lenal D+L
Dexa
Bort
D+B
[Panobinostat], nM
Dexa
Lenal D+L
Dexa
Bort
D+B
MTT studies of Panobinostat in 5 different MM cell lines: MM1S, MM1R (resistant to
MTT studies of the double and triple combinations of Panobinostat (2 nM) with Dexamethasone
Annexin V studies of the double and triple combinations of Panobinostat (7.5 nM) with
dexamethasone), U266, U266LR7 (resistant to melphalan) and U266Dox4 (resistant to
(5 nM) and Lenalidomide (1 µM) or Bortezomib (2.5 nM) in the MM1S cell line after 72 hours of
Dexamethasone (1 µM) and Lenalidomide (1 µM) or Bortezomib (3.5 nM) in the MM1S cell line
doxorubicin) after 48 hours of treatment.
treatment.
after 48 hours of treatment.
IN VIVO RESULTS
Panobinostat as a single agent is able to inhibit tumor growth and increase
Triple combinations of Panobinostat + Lenalidomide + Dexamethasone and Panobinostat + Bortezomib + Dexamethasone are more effective at inhibiting
survival in a xenograft of human plasmocytoma in CB17/SCID mice
tumor growth and increasing survival in an in vivo model of multiple myeloma as compared to their respective double combinations and the individual agents.
C
Control
Panobinostat
3000
P
)3m 2400
Ac. Histone H4
Panobinostat + Lenalidomide + Dexamethasone
Panobinostat + Bortezomib + Dexamethasone
Panobinostat + Lenalidomide + Dexamethasone
Panobinostat + Bortezomib + Dexamethasone
(m
1800
me
C
lu
C
3000
o
B
BrdU uptake
P
3000
1,0
1,0
v
1200
P
)
)
PB
3
L
D
PL
3
D
mor
m
2400
PLD
m
2400
0,8
0,8
u
600
BD
PBD
T
(m
LD
(m
l
1800
1800
a
0,6
me
0
me
iv
PL
0,6
PD
p27
lu
ival
PD
lu
v
LD
o
PD
0
20
40
60
80
o
rv
v
1200
v
1200
u
Sur
0,4
S
0,4
Days of treatment
mor
mor
L
u
600
u
600
PB
0,2
T
T
0,2
C
D
P
PD
PLD
1,0
Caspase-3
C
B
D
P
BD
PBD
0
0
0,0
0,0
0
20
40
60
80
100
0
20
40
60
80
100
120
0,8
0
20
40
60
80
0
20
40
60
80
Days of treatment
Days of treatment
Days of treatment
Days of treatment
0,6
ival
PD & PL vs any of the individuals p<0.05
PD vs any of the individuals p<0.05
rv
H2AX
PLD vs any of the doubles p<0.05
PBD vs any of the doubles p<0.05
uS 0,4
Evolution of tumor volume of immunocompromised mice (NOD-SCID) implanted with a
Evolution of survival of 120 NOD-SCID mice subcutaneously implanted with a human
0,2
Selected mice with big plasmocytomas were treated
subcutaneous human plasmocytoma (MM1S). Mice were randomized into 12 different groups
plasmocytoma (MM1S) and randomized into 12 different groups including the four
C
P
with Panobinostat and when a tumor reduction was
including the four compounds (Panobinostat, Dexamethasone, Lenalidomide and Bortezomib)
0,0
compounds (Panobinostat, Dexamethasone, Lenalidomide and Bortezomib) as single agents
0
20
40
60
80
observed,
mice
were
sacrified
to
perform
as single agents and in double and triple combinations.
and in double and triple combinations. Survival was assesed since the beginning of
Days of treatment
immunhystochemistric studies.
treatment until sacrifice due to big tumor volume (> 2 cm of diameter) or signs of toxicity.
CONCLUSIONS
· Combinations of Panobinostat + Dexamethasone + either Bortezomib or Lenalidomide are safe and display promising antimyeloma activity both in the in vitro and in vivo setting
· This study provides the rationale for the clinical development of triple combinations of these drugs to improve the outcome of MM patients
Document Outline