This study found that combining the histone deacetylase inhibitor romidepsin with inhibitors of the MAPK and PI3K pathways leads to selective toxicity in cancer cell lines with mutant Ras. The combination treatment increased apoptosis via the intrinsic apoptotic pathway in a Bax-dependent manner. Similar results were found when combining the histone deacetylase inhibitor belinostat with MAPK and PI3K inhibitors. This dual inhibition approach may help overcome resistance to histone deacetylase inhibitors in solid tumors with Ras mutations.

1. GAPDH
CONCLUSIONS
• Short-term romidepsin treatment in combination with inhibitors
of the MAPK and/or PI3K pathway is selectively toxic to cells
harboring Ras mutations
• The increased apoptosis observed in Ras mutant cancers
occurs via the intrinsic apoptotic pathway and is Bax dependent
• Dual inhibitors of the MAPK/PI3K pathway used in concert with
romidepsin are a novel combination to target Ras mutant cancers
• Romidepsin in combination with inhibitors of the MAPK or PI3K
pathway may overcome the intrinsic resistance to romidepsin in
solid tumors in the clinic
AKT and MEK inhibitors decrease AKT or ERK phosphorylation,
respectively, in cell lines with mutant and wild-type Ras
KRAS mutant (G13D) HCT-116 cells or Ras wild-type MCF-7 cells were treated with
25 ng/ml romidepsin for 6 h. They were subsequently incubated in romidepsin-free
medium for an additional 18 h; treated with 250 nM of the MEK inhibitor PD0325901
and/or 1 µM of the AKT inhibitor MK-2206 for 24 h; or treated with romidepsin in the
presence of the inhibitors for 6 hours, followed by incubation in the inhibitors alone
for an additional 18 h. Protein was then extracted and immunoblotting for the noted
proteins was performed
INTRODUCTION
The histone deacetylase inhibitor romidepsin has shown clinical efficacy in cutaneous
T-cell lymphoma and peripheral T-cell lymphoma that has not been replicated in solid
tumors. Some studies have suggested that activation of the mitogen-activated protein
kinase (MAPK) or phosphoinositide 3-kinase (PI3K) pathway can act as a resistance
mechanism for romidepsin. Oncogenic mutations in KRAS, NRAS or HRAS are
known to activate these signaling pathways. We decided to target these pathways
with MEK and AKT inhibitors concomitantly with romidepsin exposure in an effort to
overcome drug resistance. In addition, we synthesized a dual inhibitor of ERK and
PI3K and characterized its effects in combination with romidepsin
Romidepsin in combination with MK-2206 and PD0325901 elicits
selective toxicity in cells harboring mutant Ras
A series of cell lines harboring mutant or wild-type Ras were treated as outlined above
and the percent of cells staining for annexin V was calculated. The combination of
romidepsin and inhibitors of the MAPK and PI3K pathways was selectively toxic to
mutant Ras cell lines
Blocking mutant Ras signaling in the context of HDAC inhibition promotes cell death
Hanna Kozlowski, Katherine Curt, Julian Bahr, Victoria Luchenko, Agnes Basseville, Robert W. Robey, Susan E. Bates, Michael Gottesman
Laboratory of Cell Biology, NCI, NIH, Bethesda MD
The combination of romidepsin with MK-2206 and PD0325901 yields
synergistic apoptosis
KRAS mutant (G12V) SW620 colon carcinoma cells were treated with 25 ng/ml
romidepsin in combination with the MEK inhibitor PD0325901 (250 nM) or the AKT
inhibitor MK-2206 as described above. Untreated cells served as a control (C). Cells
were stained with annexin V and propidium iodide and the percent of cells staining
positive for annexin V was determined. Romidepsin in combination with the dual ERK/PI3K inhibitor D-
87503 is selectively toxic to Ras mutant cancer cell lines
KRAS mutant H460, HCT-116, A549 and SW620 cells as well as Ras wild-type 786-0
cells were left untreated ( C) or treated with 25 ng/ml romidepsin for 6 h after which
the cells were incubated in romidepsin-free medium for an additional 42 h ( RD).
Additionally, cells were treated with 5 or 10 µM D-87503 ( D-87503) for 48 h; or,
cells were treated for 6 h with romidepsin in the presence of inhibitor for 6 h, then
with inhibitor alone for an additional 42 h ( D-87503 RD). Percent of cells staining
positive for annexin V were determined and average values +/- standard deviation
are shown for at least 3 independent experiments.
Bak is dispensable yet Bax is crucial for apoptosis mediated by the
romidepsin combinations; loss of both proteins abolishes apoptosis
We have previously shown that that the mitochondrial effector proteins are important
for HDI-mediated apoptosis, leading us to examine the effects of romidepsin combined
with the various inhibitors in KRAS mutant HCT-116 parental cells or HCT-116 cells
lacking Bak, Bax or both proteins (DKO). Protein expression in the parental line and in
the knock-out lines was confirmed (inset). Treating the cell lines with the romidepsin
combinations outlined above and staining for annexin, we find Bak is dispensable for
apoptosis mediated by the romidepsin combinations, Bax is crucial, and loss of both
proteins results in near complete abolition of apoptosis. The apoptosis observed with
the romidepsin combinations therefore proceeds via the intrinsic apoptotic pathway and
is Bax-dependent.
% Annexin
Positive Cells10
0
10
1
10
2
10
3
10
4
10
0
101
10
2
10
3
10
4
SW620.024
FL1-H: FL1-Height
FL3-H:FL3-Height
7.65 60.7
13.318.3
10
0
10
1
10
2
10
3
10
4
10
0
101
10
2
10
3
10
4
SW620.017
FL1-H: FL1-Height
FL3-H:FL3-Height
0.55 4.49
4.5690.4
10
0
10
1
10
2
10
3
10
4
10
0
101
10
2
10
3
10
4
SW620.018
FL1-H: FL1-Height
FL3-H:FL3-Height
6.87 12.1
1.3779.6
10
0
10
1
10
2
10
3
10
4
10
0
101
10
2
10
3
10
4
SW620.019
FL1-H: FL1-Height
FL3-H:FL3-Height
1.79 4.02
1.4892.7
10
0
10
1
10
2
10
3
10
4
10
0
101
10
2
10
3
10
4
SW620.020
FL1-H: FL1-Height
FL3-H:FL3-Height
10.6 36.3
15.937.2
10
0
10
1
10
2
10
3
10
4
10
0
101
10
2
10
3
10
4
SW620.021
FL1-H: FL1-Height
FL3-H:FL3-Height
1.48 2.51
4.5591.5
10
0
10
1
10
2
10
3
10
4
10
0
101
10
2
10
3
10
4
SW620.022
FL1-H: FL1-Height
FL3-H:FL3-Height
9.64 24
363.3
10
0
10
1
10
2
10
3
10
4
10
0
101
10
2
10
3
10
4
SW620.023
FL1-H: FL1-Height
FL3-H:FL3-Height
1.16 5.22
1.6292
C RD 25 ng/ml
Control
(SW620)
250 nM
PD0325901
1 µM
MK-2206
PD+MK
pAKT
AKT
pERK
ERK
Bim
GAPDH
-+ - +- - + +
+ - ++- - -+RD
PD0325901
- + + ++- - -MK-2206
-+ - +- - + +
+ - ++- - -+
- + + ++- - -
H3K9ac
6h romidepsin (RD)
25 ng/ml
48 hours AKT/MEK inhibitor
Remove medium, add fresh medium
with AKT/MEK inhibitor aloneStart treatment End treatment
Treatment schema
To determine if MEK or AKT inhibitors could increase sensitivity to romidepsin, we
measured cell death by annexin assay in a panel of 14 cell lines. Cells were exposed to
25 ng/ml romidepsin for 6 h in the presence of 250 nM of the MEK inhibitor PD0325901
and/or 1 µM of the Akt inhibitor MK2206, after which romidepsin was removed and cells
were incubated with the inhibitors alone for an additional 42 h. This romidepsin treatment
more closely models the clinical setting. In treatments with belinostat
The observed apoptosis in Ras mutant cell lines is caspase-dependent
To determine if the observed cell death was caspase-mediated, we performed
combination assays on KRAS mutant SW620 and A549 cell lines in the presence of the
pan-caspase inhibitor Q-VD-OPh. Apoptosis was nearly completely abrogated when cells
were incubated with the romidepsin combinations in the presence of 10 µM Q-VD-OPh,
confirming the activation of apoptosis.
Bim
Bim
EL
L
S
HCT-116 MCF-7
7
KRAS mutant KRAS wt
Belinostat in combination with a MEK inhibitor and an AKT inhibitor is
effective in Ras mutant cancers
To verify that the observed apoptosis was not specific to romidepsin, we examined the
effects of combining a 48 h treatment of the HDI belinostat with the MEKi and/or the AKTi
in a subset of the cell lines. Belinostat at concentration of 20 and 500 nM was relatively
non-toxic to HCT-116, A549, or 786-0 cells. When combined with inhibitors of the MAPK
and PI3K pathway, increased apoptosis in the Ras mutant cell lines was observed.
C
250nMBel
MEKi
MEKiBel
AKTi
AKTiBel
MEKiAKTi
MEKiAKTiBel
C
500nMBel
MEKi
MEKiBel
AKTi
AKTiBel
MEKiAKTi
MEKiAKTiBel
C
500nMBel
MEKi
MEKiBel
AKTi
AKTiBel
MEKiAKTi
MEKiAKTiBel
0
20
40
60
80
100
%AnnexinPositive
HCT-116 A549 786-0
SW620
A549
SKMEL2
HCT-116
OVCAR5
CAL62
NCI-H460
U251
A498
HOP92
SF539
786-0
MCF-7
SN12C
KRASMutantKRASwild-type
0
100
Romidepsin
PD-0325901
MK-2206
++
++ +
++
+
15
25
50
+
+
+
+
7 22 7 58 6 43 6 82
8 28 24 55 8 51 32 78
9 28 8 38 10 58 11 77
6 36 12 54 6 55 14 71
12 17 13 41 10 31 15 63
4 20 14 36 5 49 15 61
5 16 7 22 7 23 11 54
5 9 8 15 6 15 9 34
13 13 13 18 16 20 21 33
6 13 6 16 6 19 8 32
8 14 11 18 8 23 12 23
6 9 8 11 7 25 10 16
4 8 6 8 6 9 7 12
7 10 9 11 6 10 8 10
100
50
25
15
C
Dp
PD
PDDp
MK
MKDp
PDMK
PDMKDp
C
Dp
PD
PDDp
MK
MKDp
PDMK
PDMKDp
C
Dp
PD
PDDp
MK
MKDp
PDMK
PDMKDp
C
Dp
PD
PDDp
MK
MKDp
PDMK
PDMKDp
0
20
40
60
80
100
%AnnexinPositive
10 ! M Q-VD-OPh 10 ! M Q-VD-OPh
A549 SW620
C
Dp
PD
PDDp
MK
MKDp
PDMK
PDMKDp
C
Dp
PD
PDDp
MK
MKDp
PDMK
PDMKDp
C
Dp
PD
PDDp
MK
MKDp
PDMK
PDMKDp
C
Dp
PD
PDDp
MK
MKDp
PDMK
PDMKDp
0
20
40
60
80
100
%AnnexinPositive
HCT-116 wild-type Bak-/- Bax-/- DKO
BAK
BAX
HCT-116
BAK-/-
BAX-/-
DKO
GAPDH