The histone deacetylase inhibitor, panobinostat

The histone deacetylase inhibitor, panobinostat induces apoptosis and prolongs survival
in the TH-NMYC murine model of high-risk neuroblastoma
K. Waldeck
1Peter
1,
C. Cullinane
1,
J. Shortt
2,
1,
K. Ardley
G.A. McArthur
3,
P. J. Wood
4
MacCallum Cancer Centre, Translational Research Laboratory, Melbourne, Australia, 2Peter MacCallum Cancer Centre, Gene Regulation Laboratory, Melbourne, Australia,
3Peter MacCallum Cancer Centre, Molecular Oncology Laboratory, Melbourne, Australia, 4Monash Health, Children’s Cancer Centre, Melbourne, Australia
Introduction
Deregulated acetylation of histones plays a key role in the pathogenesis of haematological as well as solid tumours by changing the chromatin structure and
consequently altering transcription of genes involved in cell cycle control, differentiation and apoptosis. Inhibitors of histone deacetylases (HDACs) can
therefore result in multiple cellular responses, and are considered to be potent inducers of cell death1. Thus, there is considerable interest in HDAC inhibition as
a potential therapeutic modality in haematological and solid tumour malignancies2. Our studies utilise the TH-NMYC murine model for neuroblastoma3 and
human neuroblastoma cell lines and confirm HDAC inhibition as a promising therapeutic target in neuroblastoma.
BMF
P a n o b in o s ta t
Actin
BIM (EL)
BIM (L)
BIM (S)
Actin
50
P v a lu e < 0 .0 0 0 1
3
2
1
0
24
hr
V e h ic le
4
r
P e r c e n t s u r v iv a l
B
0h
A
100
Panobinostat
Vehicle
BMF mRNA fold increase
(relative to 0hr)
Methods
Homozygous TH-MYCN transgenic mice underwent serial abdominal ultrasound (US) from four weeks of age until abdominal neuroblastomas greater than
50mm3 were detected. Tumour volumes were calculated followed by a survival intervention using a continuous, low dose (5mg/kg) of panobinostat, a panHDAC inhibitor for 9 weeks. US were then performed twice weekly until mice were sacrificed. Tumours were also harvested at 24 hours for western blot (WB)
and immunohistochemistry (IHC) analysis of key markers of apoptosis. In-vitro analyses of sensitivity and apoptotic response to panobinostat were also
performed in the human neuroblastoma cell lines, IMR32, SK-N-SH and the murine neuroblastoma cell line NH02A. Acetyl HH3
Panobinostat treatment
Actin
P value <0.05
0
0
25
50
75
100
125
150
Figure 4. Treatment of TH-NMYC mice with panobinostat for 24 hours resulted in
significantly increased levels of pro-apoptotic proteins BIM and BMF (shown at
both (A) protein and (B) BMF mRNA level), p<0.05.
175
D ays
Figure 1. Treatment with panobinostat significantly improved survival with 100% of THMYCN mice alive at day 63 compared with vehicle (0.0%, mean survival 7 days;
p<0.0001). One hundred days after the withdrawal of drug 88.9% of panobinostat treated
mice remained alive. IMR32
NH02A
SKNSH
A
BIM (EL)
BIM (L)
BIM (S)
V e h ic le
3
Actin
P a n o b in o s ta t
0
4
8 16 24 32 48
0
4
8
16 24 32 48
0
4
8
16 24 32 48 hours
400
B
m R N A f o ld in c r e a s e
15
200
( r e la t iv e t o 0 h r )
0
IM R 3 2
SKNSH
10
N H 02A
5
p v a lu e < 0 .0 5
60
80
Figure 2. Treatment with panobinostat caused significant and sustained tumour regression
on ultrasound. NMyc
4
8
4
0
4
8
4
0
Figure 5. Increases in the pro-apoptotic protein BIM and BMF were also observed, in
a time dependent manner, within the human and mouse panobinostat sensitive
neuroblastoma cell lines, IMR32, SK-N-SH and NH02A. (A) Western blot analysis of
pro-apoptotic protein responses following timed exposure to panobinostat (B) with
corresponding significant (p<0.05) increases in BMF mRNA at 4 hours post dose.
Key Findings
•  Low dose (5mg/kg) Panobinostat is well tolerated in the
TH-NMYC murine model when given in a continuous
dosing schedule.
•  Treatment with panobinostat prolonged survival and
caused sustained tumour regression. •  Panobinostat induces apoptosis independent of NMYC. •  These preclinical findings indicate that HDAC inhibition
is a promising therapeutic strategy, in high-risk
neuroblastoma
Panobinostat
Vehicle
H and E
4
P a n o b in o s t a t t r e a t m e n t ( h r s )
D ays
Cleaved caspase 3
2
40
2
20
2
0
8
0
0
4
T u m o u r V o lu m e ( m m )
600
Figure 3. Treatment with panobinostat for 24hr resulted in significant levels of apoptosis,
as observed by increased cleaved Caspase 3 and fragmentation of nuclei as visualized in
H and E staining. NMyc expression remained unchanged in tumour cells in response to
panobinostat.
!
References
Financial Support
1Kutko
et al, Clinical Cancer Research 2003 9(15): 5749-55
2Marks
et al, Nature Reviews 2001 1:194-202
3Weiss
et al. EMBO 1997 (16)11; 2985-2995.
You are My Sunshine (YAMS) Foundation
Kathleen Tinsley Clinical Research Fellowship

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