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Global Journal of Cancer Therapy

Richard J Rickles1, Junji Matsui3, Ping


Zhu1, Yasuhiro Funahashi2,3, Jill M
Grenier1, Janine Steiger1, Nanding
Zhao2, Bruce A Littlefield2, Kenichi
Nomoto2,3 and Toshimitsu Uenaka2*
Horizon Discovery Inc., MA, Japan
Eisai Inc., MA, Japan
3
Eisai Co., Ltd., Japan
1
2

Dates: Received: 17 October, 2015; Accepted: 03


November, 2015; Published: 04 November, 2015
*Corresponding author: Toshimitsu Uenaka, Ph.D.,
Executive Director, Production Creation Headquarter,
Oncology & Antibody Drug Strategy, CINO (Chief
Innovation Officer), E-mail:
www.peertechz.com

eertechz

Research Article

Identification of Combinatorial
Drugs that Synergistically Kill both
Eribulin-Sensitive and EribulinInsensitive Tumor Cells
Abstract
Eribulin sensitivity was examined in a panel of twenty-five human cancer cell lines representing a
variety of tumor types, with a preponderance of breast and lung cancer cell lines. As expected, the cell
lines vary in sensitivity to eribulin at clinically relevant concentrations. To identify combination drugs
capable of increasing anticancer effects in patients already responsive to eribulin, as well as inducing
de novo anticancer effects in non-responders, we performed a combinatorial high throughput screen
to identify drugs that combine with eribulin to selectively kill tumor cells. Among other observations, we
found that inhibitors of ErbB1/ErbB2 (lapatinib, BIBW-2992, erlotinib), MEK (E6201, trametinib), PI3K
(BKM-120), mTOR (AZD 8055, everolimus), PI3K/mTOR (BEZ 235), and a BCL2 family antagonist
(ABT-263) show combinatorial activity with eribulin. In addition, antagonistic pairings with other agents,
such as a topoisomerase I inhibitor (topotecan hydrochloride), an HSP-90 inhibitor (17-DMAG), and
gemcitabine and cytarabine, were identified. In summary, the preclinical studies described here have
identified several combination drugs that have the potential to either augment or antagonize eribulins
anticancer activity. Further elucidation of the mechanisms responsible for such interactions may be
important for identifying valuable therapeutic partners for eribulin.

Introduction
Eribulin mesylate, a microtubule dynamics inhibitor with a
mechanism distinct from most other anti-tubulin therapeutics, is
approved in the United States and many other countries for treatment
of certain patients with advanced or metastatic breast cancer [1,2].
Eribulin works by binding to exposed beta-tubulin subunits at the
plus ends of microtubules, where it acts through end-poisoning
mechanisms to inhibit microtubule growth and sequester tubulin
into non-functional aggregates. In mitosis, this results in G2/M cell
cycle arrest, irreversible mitotic blockade, and ultimately cell death
by apoptosis [3].
Eribulin has broad anticancer activity in a wide variety of
preclinical cancer models; as a result, numerous clinical trials of its
effectiveness under monotherapy conditions are ongoing in other
non-breast tumor types [4-9]. Although eribulin failed to show
meaningful activities in clinical trials of head and neck, pancreatic
and advanced non-small cell lung cancer [10,11], improvements in
overall survival by eribulin were reported in a Phase3 trial in advanced
soft tissue sarcoma compared with dacarbazine [12], pointing to
additional clinical uses for eribulin. Clinical trials are ongoing to
evaluate eribulin effectiveness for treatment of osteosarcoma, ovarian,
cervical, urothelium, brain metastasis, metastatic salivary gland and
pediatric cancers with the promise that additional cancer indications
will be identified.
In certain ways, the clinical experience with eribulin has been
similar to that of other chemotherapies: monotherapy benefits tend
to be limited and it is often difficult to surpass the benefits of standard

of care. Chemotherapeutic drugs are often most effective when given


in combination, in particular when synergistic killing is achieved
without additive toxicity. To date, potential combination therapies
for eribulin have been explored with only a limited number of anticancer agents. We therefore have performed an in vitro study of
eribulin combined with 34 anticancer agents in 25 different tumor cell
lines of various types, through use of a combination high throughput
screening (cHTS) platform. Our goals were to identify drugs that
might be paired with eribulin to increase clinical efficacy in metastatic
breast cancer, to identify drugs that convert eribulin non-responders
to responders, and to identify new therapeutic indications for eribulin
utilization. Several compelling synergy effects with approved and
emerging drugs were observed. The preclinical data provides insights
about future clinical development strategies.

Materials and Methods


Materials
Cell lines were purchased from European Collection of Cell
Cultures of Public Health England (A2780), Japanese Collection of
Research Bioresouces Cell Bank of the National Institute of Biomedical
Innovation (SNG-M), German Collection of Microorganisms and
Cell Cultures (KYSE-410), and American Type Culture Collection
(all other cell lines). All cell lines are included in the Cancer Cell Line
Encyclopedia [13]. Cell culture media, fetal bovine serum, and cell
culture supplements were purchased from Gibco Life Technologies
(Thermo Scientific). Chemical matter was purchased from Enzo Life
Sciences, Inc., Micro Source Discovery Systems, Sigma-Aldrich Co.
LLC, Selleck Chemicals, Sequoia Research Products Limited, and

Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identification of Combinatorial Drugs that Synergistically Kill both EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.

009

Rickles, et al. (2015)

Toronto Research Chemicals. Cell proliferation was measured by


ATP Lite 1step Luminescence Assay System (Perkin Elmer).

Methods
Cells were thawed and grown in culture media according to
vendors recommendations. Detailed methods on the combination
assay were reported in [14]. Combinations were analyzed using
Synergy Score and Loewe Volume Score, as described in [15,16] using
Horizons proprietary ChaliceTM Analyzer software.

Combination High-Throughput Screening


Cells were seeded in 384-well and 1536-well tissue culture treated
assay plates at cell densities ranging from 100 to 500 cells per well.
Twenty-four hours after cell seeding, compounds were added to
assay plates with multiple replicates. Compounds were added to cells
using a 6x6 dose matrix, formed by six treatment points (including
DMSO control) of Eribulin and each combination partner as single
agents and twenty-five combination points. Please see reference 12
for additional detail. Concentration sampling ranges were selected
after review of single agent activity of each molecule across the cell
line panel. Generally, concentrations between the EC10 and EC90
were sampled.
At the time of treatment, a set of assay plates (which do not
receive treatment) were collected and ATP levels were measured
by adding ATPLite. These Vehicle-zero (V0) plates were measured
using an EnVision Multilabel Reader (Perkin Elmer). Treated assay
plates were incubated with compound for seventy-two hours. After
seventy-two hours, treated assay plates were developed for endpoint
analysis using ATPLite. All data points were collected via automated
processes; quality controlled; and analyzed using Horizons
proprietary software.
Horizon utilizes Growth Inhibition (GI) as a measure of cell
viability. The cell viability of vehicle is measured at the time of dosing
(V0) and after seventy-two hours (V). A GI reading of 0% represents
no growth inhibition - cells treated with compound (T) and V vehicle
signals are matched. A GI 100% represents complete growth inhibition
- cells treated by compound and V0 vehicle signals are matched. Cell
numbers have not increased during the treatment period in wells with
GI 100% and may suggest a cytostatic effect for compounds reaching
a plateau at this effect level. A GI 200% represents complete death of
all cells in the culture well. Compounds reaching an activity plateau of
GI 200% are considered cytotoxic. Horizon calculates GI by applying
the following test and equation:
If T :< V0 : 100 * (1

T V0
)
V0

If T : V0 : 100 * (1

T V0
)
V V0

Where T is the signal measure for a test article, V is the vehicle-treated


control measure after seventy-two hours, and Vo is the vehicle control
measure at time zero.

Analysis of combination screening results


To measure combination effects in excess of Loewe additivity,
Horizon has devised a scalar measure to characterize the strength of
synergistic interaction termed the Synergy Score [13,14]. The Synergy

010

Score equation integrates the experimentally-observed activity


volume at each point in the matrix in excess of a model surface
numerically derived from the activity of the component agents using
the Loewe model for additivity. Additional terms in the Synergy
Score equation are used to normalize for various dilution factors used
for individual agents and to allow for comparison of synergy scores
across an entire experiment.
Loewe Volume is an additional combination model score used to
assess the overall magnitude of the combination interaction in excess
of the Loewe additivity model. Loewe Volume is particularly useful
when distinguishing synergistic increases in a phenotypic activity
(positive Loewe Volume) versus synergistic antagonisms (negative
Loewe Volume). Please see reference 13 for more detail.

Self-cross based combination screening analysis


In order to objectively establish hit criteria for the combination
screen analysis, twelve compounds were selected to be self-crossed
across the twenty-five cell line panel as a means to empirically
determine a baseline additive, non-synergistic response. The identity
of the twelve self-cross compounds was determined by selecting
compounds with a variety of maximum response values and single
agent dose response steepness. Those drug combinations which
yielded effect levels that statistically superseded those baseline
additivity values were considered synergistic. The Synergy Score
measure was used for the self-cross analysis. Synergy Scores of selfcrosses are expected to be additive by definition and, therefore,
maintain a synergy score of zero. However, while some self-cross
Synergy Scores are near zero, many are greater suggesting that
experimental noise or non-optimal curve fitting of the single agent
dose responses are contributing to the slight perturbations in the
score. Given the potential differences in cell line sensitivity to the
eribulin combination activities, we chose to use a cell-line centric
strategy for the self-cross based combination screen analysis, focusing
on self-cross behavior in individual cell lines versus global review of
the cell line panel activity. Combinations where the Synergy Score is
greater than the mean self-cross plus two standard deviations (2s)
or three standard deviations (3 s) can be considered candidate
synergies at the 95% and 99% confidence levels, respectively.

Results
Evaluation of eribulin potency using a cell-based
assay
Eribulins antiproliferative activity was assessed in a panel of
twenty-five human cancer cells lines representing a variety of tumor
types using a three-fold, ten-point dose titration of drug. Cells were
incubated with drug for 72 hours. Cellular ATP levels were quantified
as a measurement of effects on proliferation. A measurement was
performed at the time of drug addition (time zero, T0) in order to
calculate a Growth Inhibition percentage, providing information
about cytostatic and cytotoxic activities. Cell lines were designated as
sensitive to eribulin if the GI50 values were <1 nM, a concentration
deemed achievable in a clinical setting [17]. Based on the 1 nM cutoff
for drug sensitivity, 28% of the cell lines were (7/25) were deemed
to be eribulin insensitive (Figure 1). Both sensitive and insensitive
breast and lung cancer cell lines were identified. All cell lines shown

Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identification of Combinatorial Drugs that Synergistically Kill both EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.

Rickles, et al. (2015)

in Figure 1 were included in the subsequent combination screen,


with the following rationale: inclusion of eribulin sensitive cell lines
would provide models to identify drugs with the potential to increase
eribulin efficacy, while inclusion of insensitive lines would facilitate
identification of drugs capable of converting eribulin insensitive cells
to eribulin sensitive cells.

Receptor tyrosine kinase target-based cluster


analysis
The thirty-five compound enhancer library (Supplemental Table
1) was screened in combination with eribulin in the twenty-five cell
lines described above in Figure 1. In order to objectively establish hit
criteria for the combination screen analysis, twelve compounds were
selected to be self-crossed across the twenty-five cell line panel as a
means to empirically determine a baseline additive, non-synergistic
response. The identity of the twelve self-cross compounds was
determined by selecting compounds with a variety of maximum
response values and single agent dose response steepness. Those drug
combinations which yielded effect levels that statistically superseded
those baseline additivity values were considered synergistic.
A summary matrix view heat map of combination drug Synergy
Scores which exceed the cell line specific self-cross thresholds at
2s above the mean is shown in Supplemental Figure 1. Using this
strategy, 19.7% of the combinations exhibited some synergistic
activity at the 95% confidence level. Using a more stringent criteria
of 3 cutoff (99% confidence), synergy was observed for 12.5% of the
combinations (Supplemental Figure 2).
The 35 compound enhancer library utilized contains some
redundancy in terms of targets and pathway inhibitors. Whenever
possible, target information was used to cluster similarly annotated
enhancers across the cell line panel, and the Loewe Excess values
for Growth Inhibition matrices with high-to-moderate synergy
scores were individually reviewed to evaluate combination activities.

As an example of enhancer library mechanistic redundancy,


the combination screen contained three drugs known to inhibit
ErbB1/ErbB2 (EGFR/HER2) in cell-based assays: lapatinib, BIBW2992 and erlotinib [18-21]. BIBW-2992 exhibited the best breadth
of combination activity. The mechanism of action of BIBW-2992
(irreversible inhibition) and higher potency for ErbB1 and ErbB2
may explain the greater breath-of-activity. Alternatively, synergies
observed with BIBW-2992 but not lapatinib or erlotinib may be
the result of unique polypharmacy with BIBW-2992 inhibiting
secondary targets not affected by the other ErbB1/ErbB2 inhibitors.
Representative Growth Inhibition and Loewe Excess dose matrices
for some of the lapatinib combinations are shown in Figure 3 with
examples of erlotinib and BIBW-2992 combination activities shown
in Supplemental Figures 3, 4. Low micromolar concentrations of
lapatinib and erlotinib have been observed in pharmacokinetic
studies [18,19] suggesting the potential for reproducing the observed
preclinical synergy here in a clinical setting.

PI3K pathway inhibitors


Dysregulation of the PI3K pathway can transform cells by virtue
of constitutive activation and ultimately, stimulation of cellular
proliferation and suppression of pro-apoptotic signaling. Four PI3K
pathway inhibitors were included in the screen. AZD8055 is a potent,
selective, and orally bioavailable ATP-competitive mTOR kinase
inhibitor (TORC1 and TORC2); everolimus, an allosteric mTOR
(TORC1) inhibitor; BEZ235, a dual pan-PI3K/mTOR inhibitor; and
35.00
25.00
15.00
5.00
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

Ovarian

Endometrial
Liver
Esophageal
Head+Neck
Kidney
Bladder
Pancreas
Prostate
Melanoma
Fibrosarcoma
Stomach

Lung

Breast

Hep G2
T24
MDA-MB-231
NCI-H460
786-0
T47D
A375
A2780
KATOIII
FaDu
NCI-H1650
A549
MCF7
NCI-H526
SNG-M
NCI-H522
NCI-H69
MDA-MB-436
KYSE-410
PC-3
MIA PaCa-2
HT-1080
SK-BR-3
SK-OV-3
MDA-MB-468

1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

MCF7
MDA-MB-231
MDA-MB-436
MDA-MB-468
SK-BR-3
T47D
A549
NCI-H1650
NCI-H460
NCI-H522
NCI-H526
NCI-H69
A2780
SK-OV-3
SNG-M
Hep G2
KYSE-410
FaDu
786-0
T24
MIA PaCa-2
PC-3
A375
HT-1080
KATO III

Eribulin GI5 0 (nM )

35.00
25.00
15.00
5.00

Eribulin GI 5 0 (nM )

The strategy of using target or pathway cluster profiles with visual


inspection of matrices provides additional evidence linking a
particular target with combination activity, and helps to highlight
subtle synergies that might otherwise be overlooked or insufficiently
revealed due to steep single agent dose response curves. A Synergy
Score heat map for select target/pathway clusters is displayed in
Figure 2, with cell lines clustered first according to eribulin sensitivity/
insensitivity then further demarcated based on tumor type.

Figure 1: Assessment of eribulin activity in twenty-five cell lines. Bar graph display of GI50 values based on relative sensitivity (Waterfall plot, left) or after
clustering cell lines based on tumor type (right). Cells were exposed to eribulin for 72 hours. The assay endpoint was measurement of ATP (as a surrogate for effects
on proliferation). The median GI50 for the twenty-five cell line panel is 0.51 nM.

011

Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identification of Combinatorial Drugs that Synergistically Kill both EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.

Rickles, et al. (2015)

PI3K Pathway

MEK

NCI-H522

NCI-H69

A2780

SNG-M

KYSE-410

MDA-MB-231

T47D

NCI-H460

Hep G2

786-0

T24

A375

9.00

6.21

6.78

6.74

6.28

3.02

5.01

18.61

7.59

9.83

16.05

7.77

6.22

5.05

4.20

1.45

4.90

12.25

17.59

8.52

11.31

8.63

12.25

3.24

12.15

1.26

2.96

3.25

4.90

2.49

3.91

5.50

3.94

4.38

7.25

23.98

1.49

2.10

1.18

4.36

0.96

2.99

7.77

5.48

7.34

6.49

6.74
11.77

KATOIII

PC-3

FaDu

HT-1080

NCI-H1650

1.80

3.14

MIA PaCa-2

A549

1.31

Erlotinib

SK-OV-3

SK-BR-3

BIBW-2992

NCI-H526

MDA-MB-468

ErbB1/ErbB2

Eribulin +

MDA-MB-436

Target

Eribulin-Insensitve Cell Lines

MCF7

Eribulin-Sensitive Cell Lines

Lapatinib

7.86

1.77

9.33

2.35

1.65

2.20

0.43

1.89

4.55

6.87

4.80

8.69

9.15

26.61

0.63

0.47

1.36

3.25

0.76

3.11

9.85

7.68

17.46

14.14

AZD 8055

11.84

5.04

8.60

1.13

7.71

6.53

1.39

2.96

8.19

17.12

7.62

5.44

9.68

9.38

1.53

8.79

10.47

6.44

4.14

7.65

10.15

1.70

2.85

4.48

5.37

BEZ235

8.03

5.32

16.92

1.32

6.78

5.92

2.86

2.34

7.90

10.80

4.43

12.44

9.16

18.70

2.36

8.18

9.60

4.34

7.28

13.32 12.30

10.10

4.16

6.75

8.36

BKM-120

6.12

2.87

6.05

1.13

5.38

2.82

1.20

3.71

5.47

10.76

3.27

7.03

4.86

7.72

0.75

3.02

8.11

2.79

2.10

7.17

5.99

3.22

2.49

2.34

1.45

Everolimus

4.69

2.78

11.76

2.56

1.56

5.84

1.50

4.05

7.14

6.97

4.84

4.14

5.08

5.71

1.02

4.24

9.81

3.92

2.88

7.07

1.97

1.85

1.60

1.56

4.42

Trametinib

1.32

3.98

10.18

0.50

5.63

3.66

0.26

0.87

2.09

14.55

4.80

0.71

4.10

4.76

3.87

0.88

0.72

1.69

14.92

1.58

9.31

10.41

3.65

2.90

1.10

E6201

0.71

1.72

2.19

0.00

7.06

4.05

1.02

1.61

2.42

19.08

0.36

1.19

3.32

7.22

12.64

0.23

1.26

1.93

11.75

2.35

6.26

18.64

2.78

2.01

1.22

Figure 2: Synergy Score heat map of selected compounds screened in combination with eribulin and clustered based on target or pathway specificity.
Twenty five cell lines were screened, shown as vertical columns labeled across the top and grouped first based on eribulin sensitivity/insensitivity then further
clustered based on tumor type (breast cancer, blue; lung cancer, orange; ovarian cancer, green; single representative cancers, not highlighted; see Figure 1 for
information on other cancer types). Each row lists one compound screened in combination with eribulin, with compounds clustered based on target or pathway
specificity. Synergy Scores are shown, with higher scores highlighted in increasingly darker shades of red. For this analysis, a Synergy Score cut-off of 4.36 was
chosen based on visual inspection of dose matrices and Loewe Excess matrices above and below this cut-off score.

75 126 147

50

80

94 131

.1

Eribulin(E7389) (uM)

18

29

22

13

23

-1

19

-11

-14

20
4

1.4e-4 4.1e-4 .0012 .0037

82

88

82

70

11

48

78

85

82

76

-3

33

48

76

71

86

25

34

58

73

94

19

21

42

56

73

.011

Eribulin(E7389) (uM)

15

10

11

-3

15

25

-7

-1

-12

74

68

71

78

-6

52

72

67

79

-15

-3

38

63

46

78

-15

-1

35

33

-6

-6

-8

93 135 160 142 174

73
0

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

74

94 130 129 146

-1

53

85

88 102

1.4e-4 4.1e-4 .0012 .0037

Eribulin(E7389) (uM)

.0012 .0037

.011 .033

Growth Inhibition (%) Vol=7.77(.5)


Chi2=270

Lapatinib (uM)

9.9
3.3
1.1
.37
.12

-1

.1

.0012 .0037

.011 .033

39

41

53

59

54

43

43

34

17

-2

11

33

49

39

21

-3

20

23

31

11

16

15

13

24

-1

-4

.1

13

-4

.0012 .0037

.011 .033

4
.1

Eribulin(E7389) (uM)

Dose MatrixNone | 100 | 786-0 |


ATP Lite | 72h |

DT-1308660

.011

-17

786-0
12

82

DT-1308669

51
6

1.4e-4 4.1e-4 .0012 .0037

.011

Eribulin(E7389) (uM)

Loewe ExcessNone | 100 | 7860 | ATP Lite | 72h |

DT-1308661

91 167 173 189 191 197


10

78

89 113 134 117

60

97 118 129 113

36

78 102 113 129

-2

25

44

76

94 126

27

32

42

71 112

.0012 .0037

.011 .033

.1

Eribulin(E7389) (uM)

N=2

49

12

Loewe ExcessNone | 100 | Hep


G2 | ATP Lite | 72h |

Growth Inhibition (%)

26

Eribulin(E7389) (uM)

9.9

14

N=2
Growth Inhibition (%)

9.9
3.3
1.1
.37
.12
0

Lapatinib (uM)

Chi2=66
Growth Inhibition (%) Vol=4.7(.48)

9.9
3.3
1.1
.37
.12

-9

-6

DT-1308661

74

78

91

89

92

49

44

42

45

13

44

65

61

45

23

51

48

31

27

13

17

24

12

23

15

-10 -10

.011 .033

.1

0
1
0
-2
0

.0012 .0037

Eribulin(E7389) (uM)

A375
Dose MatrixNone | 100 | A375 |
ATP Lite | 72h |

59

70

45

69

96

92

65

36

44

81

84

57

36

-4

39

65

30

19

-7

-6

-2

42

11

21

-7

-5

-1

.0012 .0037

.011 .033

1
.1

Eribulin(E7389) (uM)

66

86

97 135 142 166

Loewe ExcessNone | 100 | A375


| ATP Lite | 72h |

22

72

90 105 121 118

63

87 110 122 123

41

77

96 106 115

-3

38

52

75

96 119

20

30

53

74

1.4e-4 4.1e-4 .0012 .0037

96
.011

Eribulin(E7389) (uM)

DT-1308663

19

29

66

64

74

-1

33

38

40

43

26

43

50

51

44

31

25

43

39

29

23

-3

23

20

19

18

-1

-2

-4

9.9

41

3.3

37

1.1

DT-1308663

N=2

DT-1308662

1.4e-4 4.1e-4 .0012 .0037

27
5

Growth Inhibition (%) Vol=9.22(.44)


Chi2=80

Loewe ExcessNone | 100 | T24 |


ATP Lite | 72h |

Growth Inhibition (%)

Lapatinib (uM)

91

17

69

3.3

-1

9.9

63

22

91

3.3

13

.011 .033

41

88

1.1

-4

.0012 .0037

36

88

.37

-6

16

86

.12

76 111

13

1.1

Lapatinib (uM)

54

-6

53

98 149 168 164 175

-1

19

75 115 117 128 135 131

.37

22

Growth Inhibition (%) Vol=11.3(.55)


Chi2=150

-6

38

12

90

12

Eribulin(E7389) (uM)

.12

64

28

DT-1308669

Lapatinib (uM)

53

9.9

.12

-7

58

109 149 183 185 184 187


90 109 170 165 167 174

36

3.3

94 109

29

Growth Inhibition (%) Vol=13.1(.41)


Chi2=270

-9

1.1

79

11

9.9

-1

.37

40

-1

3.3

-1

.12

24

1.1

45

Lapatinib (uM)

.37

-4

44

.37

30

83 101 124 126

N=2

44

61

.12

-8

Eribulin(E7389) (uM)

Growth Inhibition (%)

1.1

9.9
3.3

73 107 127 135 126

DT-1308667

24

DT-1308660

38

Hep G2

Loewe ExcessNone | 100 | FaDu


| ATP Lite | 72h |

T24

Eribulin(E7389) (uM)
Dose MatrixNone | 100 | Hep G2
| ATP Lite | 72h |

Dose MatrixNone | 100 | FaDu |


ATP Lite | 72h |

N=2

29

9.9

-41 -42 -24

9.9

N=2

27

DT-1308662

98 105 127 140 135

Eribulin(E7389) (uM)

12

Eribulin(E7389) (uM)

22

.1

Loewe ExcessNone | 100 | SNGM | ATP Lite | 72h |

.011

15

-4

Growth Inhibition (%) Vol=7.01(.44)


Chi2=20

66

1.4e-4 4.1e-4 .0012 .0037

37

Growth Inhibition (%) Vol=11.1(.35)


Chi2=190

32

64

9.9

51

3.3

83 136 150

37

1.1

63

14

-4

.37

14

DT-1308666

36

Dose MatrixNone | 100 | T24 |


ATP Lite | 72h |

61

.011 .033

FaDu
34

Eribulin(E7389) (uM)

.0012 .0037

18

.12

20

.1

36

48

Lapatinib (uM)

64 113 148 149

Loewe ExcessNone | 100 | KYSE


-410 | ATP Lite | 72h |

91 114

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

.37

97

55

53

42

Growth Inhibition (%)

78

49

65

9.9

16

3.3

80

38

36

3.3

86 135 145 146

1.1

79 105 115

37

18

1.1

-29 -14

57

.37

56

-3

99 120

31

.37

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Lapatinib (uM)

9.9

N=2

79

53

.12

19

36

.12

28

-2

53

67

Lapatinib (uM)

-1

90 137 152 151

24

84 107 121 118

27

-4

74

Lapatinib (uM)

91 116 139

49

N=3-4

-22

Growth Inhibition (%) Vol=4.13(.49)


Chi2=99

57

-10

-4

Loewe ExcessNone | 100 | NCIH460 | ATP Lite | 72h |

DT-1308664

Growth Inhibition (%)

3.3

37

98 135 130 121

9.9

12

1.1

.37

48

65

3.3

23

.37

96 143 154

-2

-4

1.1

21

.12

74

94 100 119 111 120

.37

Lapatinib (uM)

69

-2

.12

-7

-15 -27

1.1

62

14

DT-1308667

62

Eribulin(E7389) (uM)

Growth Inhibition (%)

9.9
3.3

91 107 145 160

.12

Lapatinib (uM)

73

74

11

13

96 100 103 111 142

-4

-2

24

22

29

Lapatinib (uM)

-1

DT-1308666

17

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

KYSE-410
181 186 193 192 193 185

Growth Inhibition (%) Vol=5.87(.48)


Chi2=37

16

Dose MatrixNone | 100 | KYSE410 | ATP Lite | 72h |

10

9.9

22

Eribulin(E7389) (uM)

12

3.3

-1

.011 .033

23

1.1

-15

.0012 .0037

16

.37

-9

-2

.12

14

Lapatinib (uM)

-13

9.9
3.3

N=2

54

.1

19

.12

.011 .033

43

1.1

90 108 123 144 153


.0012 .0037

40

.37

38

.12

93 115 132 136 156

65

Lapatinib (uM)

13

30

DT-1308664

SNG-M

.37

92 121 110 139 160

25

Dose MatrixNone | 100 | SNG-M


| ATP Lite | 72h |

DT-1308670

Growth Inhibition (%)

9.9
3.3
1.1

93 133 130 138 158

.12

Lapatinib (uM)

25

98 127 122 136 159

-1

Eribulin(E7389) (uM)

Loewe ExcessNone | 100 |


A2780 | ATP Lite | 72h |

DT-1308670

30

Eribulin(E7389) (uM)

A2780

Dose MatrixNone | 100 | A2780


| ATP Lite | 72h |

86 154 174 176 180 191

.1

90 150 164 165

-19

Lapatinib (uM)

Eribulin(E7389) (uM)

.011 .033

.0012 .0037

54

29

34

.1

65 102 140 169 165

50

18

80 113 156 167 174

10

Growth Inhibition (%)

-3

3.3

-21 -16

1.1

.37

31

.12

23

N=2

9.9

17

-7

64

Lapatinib (uM)

.011 .033

-22 -14

59

.0012 .0037

-20 -20

-1

11

NCI-H460

Dose MatrixNone | 100 | NCIH460 | ATP Lite | 72h |

DT-1308665

N=2

-7

-4

86 128 154 170 174

9.9

93 115

19

34

3.3

81

21

91 139 172 179 181

1.1

76

22 -19 -26 -15

56

.37

69

54

.12

31

89 120

18

120 173 187 192 194 196

Lapatinib (uM)

82

-5

Loewe ExcessNone | 100 | MDAMB-468 | ATP Lite | 72h |

DT-1308665

Lapatinib (uM)

77 100 114

75

-9

-3

Growth Inhibition (%) Vol=2.46(.26)


Chi2=330

77

68

65

Growth Inhibition (%) Vol=2.33(.51)


Chi2=6.1

75

-4

59

3.3

.37

19

52

1.1

82 102 118

32

.37

71

27

.12

73

Lapatinib (uM)

1.1

24

92 114 127 151

Dose MatrixNone | 100 | MDAMB-468 | ATP Lite | 72h |

DT-1308668

Growth Inhibition (%)

9.9
3.3

88

.12

Lapatinib (uM)

46

132 156 161 181 188 194

MDA-MB-468

Loewe ExcessNone | 500 | MCF7


| ATP Lite | 72h |

DT-1308668

Growth Inhibition (%)

MCF7

Dose MatrixNone | 500 | MCF7 |


ATP Lite | 72h |

.011

Eribulin(E7389) (uM)

Figure 3: Representative dose matrices for eribulin x lapatinib combination activity. Selected dose matrix pairs for eribulin in combination with lapatinib are
shown in cell lines MCF7, MDA-MB-468, NCI-H460, A2780, SNG-M, Hep G2, KYSE-410, FaDu, 786-0, T24 and A375. For each cell line, Growth Inhibition values
(left matrix) and Loewe Excess values (right matrix) are shown. The Growth Inhibition dose matrix values are determined by measurement of ATP levels with a
T0 measurement (at time of drug addition) performed at the whole well level in order to distinguish cytostasis from cytotoxicity. Values highlighted in medium red
are those approximating GI100% and are indicative of compounds/combinations being cytostatic; values with highlighting closest to black are those approximating
GI200% and indicate complete killing (cytotoxic effect).

BKM-120, a pan-PI3K inhibitor. As shown in Figures 2, 4, one or


more of these inhibitors are synergistic in combination with eribulin
in the majority of cell lines in the panel. For a subset of cell lines,
synergy is observed with all four PI3K pathway inhibitors. The
breadth of activity observed in the cell line panel and for the various
PI3K pathway inhibitors highlight the importance of PI3K signaling
for tumor cell proliferation and/or survival upon eribulin exposure

012

(Figure 4). Combination activity is selective (for example, little or no


combination activity in SK-BR-3, NCI-H552, NCI-H526, Mia PaCa2, and 786-0) pointing to specific genetic determinants in responsive
cell lines as being important for combination activity.

MEK inhibitors
Two MEK inhibitors (E6201 and trametinib) were screened in

Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identification of Combinatorial Drugs that Synergistically Kill both EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.

Rickles, et al. (2015)

HT-1080

T47D

NCI-H460

Hep G2

1.39

2.96

8.19

17.12

7.62

5.44

9.68

9.38

1.53

8.79

10.47

6.44

4.14

7.65

10.15

1.70

2.85

4.48

5.37

5.92

2.86

2.34

7.90

10.80

4.43

12.44

9.16

18.70

2.36

8.18

9.60

4.34

7.28

13.32 12.30 10.10

4.16

6.75

8.36

BKM-120

6.12

2.87

6.05

1.13

5.38

2.82

1.20

3.71

5.47

10.76

3.27

7.03

4.86

7.72

0.75

3.02

8.11

2.79

2.10

7.17

5.99

3.22

2.49

2.34

1.45

Everolimus 4.69

2.78

11.76

2.56

1.56

5.84

1.50

4.05

7.14

6.97

4.84

4.14

5.08

5.71

1.02

4.24

9.81

3.92

2.88

7.07

1.97

1.85

1.60

1.56

4.42

Dose Matrix | None | 100 | HT1080 | ATP Lite | 72h

103 153 173 185 186 186

139 148 165 163 170 180

.012 .037 .11

103 155 164 167 176 175

95 128 130 167 175 188

58 105 131 134 156 168

.011

.0012 .0037

.011 .033

.1

91 104

1.4e-4 4.1e-4 .0012 .0037

40

72 105 157 181

22

57

.011

96 151 180

Eribulin(E7389) (uM)

Eribulin(E7389) (uM)

Eribulin(E7389) (uM)

Eribulin(E7389) (uM)

Dose Matrix | None | 100 | MDA


-MB-468 | ATP Lite | 72h

Dose Matrix | None | 500 | NCIH69 | ATP Lite | 72h

Dose Matrix | None | 100 |


A2780 | ATP Lite | 72h

Dose Matrix | None | 100 | FaDu


| ATP Lite | 72h

Dose Matrix | None | 100 | HT1080 | ATP Lite | 72h

104 121 156 165 168 164

164 170 171 171 175 174

122 161 169 181 177 185

84 106 142 141 158 164

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Eribulin(E7389) (uM)

1.4e-4 4.1e-4 .0012 .0037

.011

Eribulin(E7389) (uM)

54

89

92

Dose MatrixNone | 100 | FaDu |


ATP Lite | 72h |

74 111 147 151 169 179

44

58

73 101 132 136

42

59

75

95 108 147

34

43

72

89

37

57

68

89 103 128

33

35

77

92

93 128

-1

53

85

88 102

30

97 136 136 159 173

14

90 123 123 143 161

90 111 130 147 159

90 108 123 144 153

.0012 .0037

.011 .033

92 150 181

96 141 178

66

-5
0

.011

Dose MatrixNone | 100 | A2780


| ATP Lite | 72h |

DT-1308899

N=1-2

99

.66

1.4e-4 4.1e-4 .0012 .0037

.025 .074 .22

91 111

70

30

81

35

BKM-120 (uM)

60

Eribulin(E7389) (uM)

62 110 148 141 163 180

Growth Inhibition (%)

2
.66
.025 .074 .22

BKM-120 (uM)

N=2

2
.66
.025 .074 .22

BKM-120 (uM)

Growth Inhibition (%)

44

8
12

.1

Eribulin(E7389) (uM)

1.4e-4 4.1e-4 .0012 .0037

.011

Eribulin(E7389) (uM)

63

92 145 175

25

56

89 124 163

82 112 144 177 185

54

73

50

70 104 145 173 184

53

74

96 153 171 182

43

54 122 150 168

97 144 169 180

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Eribulin(E7389) (uM)

99 102 90

.0012 .0037

.011 .033

67

Growth Inhibition (%)

.7
.026 .078 .23
.0086

N=2

BEZ235

.1

DT-1308718

94 129 127 141 128 132


92 106 127 120 104

19

79

13

57 108 113 107 67

13

63

98 106 103 74

63

90 101 98

99 123 104 83

.0012 .0037

.011 .033

72

BKM-120

.1

Eribulin(E7389) (uM)
Dose MatrixNone | 100 | T47D |
ATP Lite | 72h |

DT-1308900

76 107 147 175 175

60

32

Dose MatrixNone | 100 | HT1080 | ATP Lite | 72h |

48

83 100 115 118 95

Dose Matrix | None | 100 |


T47D | ATP Lite | 72h

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

51

55

Eribulin(E7389) (uM)

DT-1308895

98 142

26

.017 .05

66 121 133 135

.1

-3

99 114

77 119 174 180

6.2e-4 .0019 .0055

12

84

59

Everolimus (uM)

86 100 128

62

99 145 161 171 186

19

97 103 125 140 142 132

Eribulin(E7389) (uM)

DT-1308722

85

65 134 150 145

75

37

N=2

72 134 151 155

21

43

Growth Inhibition (%)

76 139 146 160

26

14

9
18

.017 .05

33

14

.011 .033

95 113 162

6.2e-4 .0019 .0055

20

90 100 109 129 143


.0012 .0037

84

Eribulin(E7389) (uM)

N=2

N=2

86 151 163 164

91 106 110 130 149

66

Everolimus (uM)

90 150 164 165

53

94 114 123 138 159

23

54

56 106 150 162 166

23

7.6e-6 2.3e-5 6.8e-5 2.1e-4 6.2e-4

7.6e-6 2.3e-5 6.8e-5 2.1e-4 6.2e-4

91 154 171 168

Everolimus (uM)

81

72 127 157 172 177

Growth Inhibition (%)

7.6e-6 2.3e-5 6.8e-5 2.1e-4 6.2e-4

84 130 163 185 186

DT-1308896

35

.011

Dose MatrixNone | 500 | NCIH69 | ATP Lite | 72h |

DT-1308898

25

62 126 143 147

N=2

Dose MatrixNone | 100 | MDAMB-468 | ATP Lite | 72h |

39 117 162 176 185 183

27

1.4e-4 4.1e-4 .0012 .0037

Eribulin(E7389) (uM)

Eribulin(E7389) (uM)

64 119 158 185 187 187

62 136 135 153

21

DT-1308721

Growth Inhibition (%)

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

20

30 109 130 132 151 166

Everolimus (uM)

64 106 136 142

76 133 151 156

75 125 148 147 160 179

45

98 148 158 160

41

Growth Inhibition (%)

63 110 137 142

58

16

N=2

15

34

DT-1308720

Growth Inhibition (%)

97 143 132

.66

59

93 140 160 167 168

.025 .074 .22

72 148 146 155

74

16

N=2-3

13

-22 29

DT-1308719

BKM-120 (uM)

99 142 150 163

Growth Inhibition (%)

.025 .074 .22

.66

BKM-120 (uM)

DT-1308717

54

107 120 134 150 156 145

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Eribulin(E7389) (uM)

18

.33

85 112 173 187

BEZ235 (uM)

62

.012 .037 .11

99 104 153 175 184

30

84

.012 .037 .11

52

.0014 .0041

-4

77

N=1-2

66 105 97 153

BEZ235 (uM)

33

Growth Inhibition (%)

N=2

24

98 140 160 161

104 123 141 146 150 141

1.4e-4 4.1e-4 .0012 .0037

89 108 115 133 159

73 105 102 142 172

DT-1308725

100 125 139 138 145 147

.66

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

58 126 144 146

94 134 127 139 165

.33

15

33

72

49

BEZ235 (uM)

N=2

N=1-2

.012 .037 .11

83 140 147 159

67

82 115 126 163 179 188

.1

.025 .074 .22

33

.012 .037 .11

82 120 143 158

16

89 109 143 147 166 180

81 102 125 152 170 176

66

Dose Matrix | None | 100 |


T47D | ATP Lite | 72h

DT-1308728

DT-1308723

100 123 159 170 178 170

61

85 147 158 163

99 138 159 163 173 180

Growth Inhibition (%)

32

BEZ235 (uM)

88 134 168 165

52 112 155 166 165

13

33

57

DT-1308727

Growth Inhibition (%)

25

.0014 .0041

66 105 146 173 176

BEZ235 (uM)

22

103 113 148 164 169 174

.0014 .0041

48 103 149 173 182 182

DT-1308726

Growth Inhibition (%)

93 127 173 184 191 187

.011 .033

N=1-2

Dose Matrix | None | 100 | FaDu


| ATP Lite | 72h

71 100 100 94
.0012 .0037

AZD 8055

Eribulin(E7389) (uM)

Growth Inhibition (%)

Dose Matrix | None | 100 |


A2780 | ATP Lite | 72h

96 109 106 79

N=2

Dose Matrix | None | 500 | NCIH69 | ATP Lite | 72h

76

Growth Inhibition (%)

Dose Matrix | None | 100 | MDA


-MB-468 | ATP Lite | 72h

80 103 112 106 92

35

DT-1308897

32

84 106 129 106 102

31

74 105 111 118 96

15

74 106 120 118 81

36

75 108 117 114 90

29

83 112 111 104 90

51

88 104 94

.0012 .0037

.011 .033

73

N=1-2

Eribulin(E7389) (uM)

56

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Eribulin(E7389) (uM)

81 104 116 113 128 111

Growth Inhibition (%)

86 135 174

AZD 8055 (uM)

64

14

.011

Eribulin(E7389) (uM)

BKM-120 (uM)

95 102

74 102 146 179

89

1.4e-4 4.1e-4 .0012 .0037

38

99 109 130 132 129 125

.017 .05

54

11

100 128 126 141 141 127

6.2e-4 .0019 .0055

.1

83 109 171 184

Everolimus (uM)

94 134

92 140 174 187

52

80

70

32

N=2

56

57

Growth Inhibition (%)

25

.7
.026 .078 .23

AZD 8055 (uM)

N=2

97 129

.0014 .0041

.011 .033

94

.0086

91 104 108 128 147


.0012 .0037

58

.011

Growth Inhibition (%)

.7
.026 .078 .23
.0086

AZD 8055 (uM)

N=2
Growth Inhibition (%)

.7
.026 .078 .23
.0086

N=2-3

AZD 8055 (uM)

Growth Inhibition (%)

AZD 8055 (uM)

61 130 140 150

1.4e-4 4.1e-4 .0012 .0037

74 103 130 166

15

Eribulin(E7389) (uM)

BEZ235 (uM)

.012 .037 .11

21

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

54 110 138 143 152 177

39

10

Eribulin(E7389) (uM)
DT-1308724

Everolimus (uM)

87 147 161 162

25

N=2

64 106 136 150

46

76 110 134 152 170 179

DT-1308695

Growth Inhibition (%)

52

19

90 126 150 157 173 182

91 110 147 176 185

N=2

69 107 135 154

62 100 153 163 164

77

Growth Inhibition (%)

38

84 123 159 165 168

39

87 117 142 155 179 184

N=2

57

94 106 145 162

Growth Inhibition (%)

60 109 152 142

66

.7

71 132 159 162

26

73 106 146 144 178

56

.026 .078 .23

55

14

73

.0086

17

97 137 158 167 177 184

DT-1308706

103 152 166 170 177 178

104 104 143 163 162 166

N=1-2

132 132 149 159 164 169

62 100 157 180 174

T47D
Dose Matrix | None | 100 |
T47D | ATP Lite | 72h

Dose Matrix | None | 100 | HT1080 | ATP Lite | 72h

DT-1308703

AZD 8055 (uM)

61 121 174 184 179

26

HT-1080

Dose Matrix | None | 100 | FaDu


| ATP Lite | 72h

DT-1308693

Growth Inhibition (%)

47

DT-1308699

FaDu

FaDu

Dose Matrix | None | 100 |


A2780 | ATP Lite | 72h

DT-1308694

SNG-M

A2780

A2780

Dose Matrix | None | 500 | NCIH69 | ATP Lite | 72h

.026 .078

NCI-H69

Dose Matrix | None | 100 | MDA


-MB-468 | ATP Lite | 72h

9.6e-4 .0029 .0086

MDA-MB-468

A375

PC-3

6.53

6.78

T24

NCI-H526

7.71

1.32

786-0

NCI-H522

1.13

16.92

KATOIII

NCI-H1650

8.60

5.32

MIA PaCa-2

A549

5.04

8.03

KYSE-410

SK-BR-3

11.84

BEZ235

SK-OV-3

MDA-MB-468

AZD 8055

NCI-H69

MDA-MB-436

PI3K
Pathway

Eribulin +

MCF7

Target

Eribulin-Insensitve Cell Lines


MDA-MB-231

Eribulin-Sensitive Cell Lines

Everolimus

.1

Eribulin(E7389) (uM)

Figure 4: Synergy Score heat map and representative Growth Inhibition dose matrices for eribulin x PI3K pathway combination activities. Select pairs
of dose matrix plots for eribulin in combination with PI3K pathway inhibitors are shown for cell lines MDA-MB-468, NCI-H69, A2780, HT-1080 and T47D. Growth
Inhibition dose matrices for AZD8055 (mTOR TORC1/2 kinase inhibitor), BEZ235 (pan-PI3K/mTOR inhibitor), BKM-120 (pan-PI3K inhibitor) and everolimus (mTOR
TORC1 inhibitor) are shown.

combination with eribulin (Figure 2); selected Growth Inhibition


and Loewe Excess dose matrices are shown in Figure 5. There is good
concordance for both inhibitors; when synergy is observed with
one of the MEK inhibitors, it is most often observed with the other.
MEK combination activity was strongest in MDA-MB-231, A2780
and Hep G2. For some cell lines, synergy is observed when eribulin
is combined with either PI3K pathway inhibitors or MEK inhibitors
(for example, FaDu, NCI-H460 and A2780). For other cell lines,
(MCF7, NCI-H69, KYSE-410, HT-1080 and T47D), synergies are
PI3K pathway-specific, with no synergies seen for MEK inhibitors.

BCL-2 Inhibition
ABT-263 (Navitoclax) is a potent Bcl-xL, Bcl-2, and Bcl-w
inhibitor currently in multiple clinical trials as a monotherapy or in
combination with approved drugs. Strong synergies with eribulin
and good breadth of activity was observed with ABT-263, in both
eribulin-sensitive and insensitive cell lines (Figure 6). Strong synergy
was observed in most but not all of the cell lines, suggesting that an

013

eribulin x ABT-263 combination is not non-selectively synergistically


toxic.

Antagonistic drug pairings


As described in Materials and Methods, Loewe Volume values can
be used to gauge both synergies and antagonisms. A Loewe Volume
heat map is shown in Figure 7 for 17-DMAG, cytarabine, topotecan,
and gemcitabine, with greater negative values (antagonism)
highlighted in increasingly darker shades of blue and increasing
positive values (synergies) highlighted in increasingly darker shades
of red. Also shown are representative combination dose matrices
for selected antagonistic activities observed. The 17-DMAG Loewe
Volume combination activity pattern is complex: for 6 cell lines (red
shaded), combinations with eribulin are synergistic, while for 4 cell
lines, antagonism was observed.

Discussion
Eribulin has broad anticancer activity in a wide variety of

Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identification of Combinatorial Drugs that Synergistically Kill both EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.

Rickles, et al. (2015)

-6 -13 -10 -1

46

47

69

86

.0012 .0037

.1

10
3.3
1.1
.37
.12

ANDI(E6201) (uM)

-1

-5

.011 .033

.1

91 104 108 128 147


.0012 .0037

.011 .033

DT-1308808

Dose MatrixNone | 100 | A2780


| ATP Lite | 72h |

27

57

57

64

57

-2

18

30

27

36

45

-5

12

22

20

24

34

.025 .074 .22

95 139 156 157 167 176

.66

21

20

18

67 108 135 140 157 172

14

11

22

.0027 .0082

Loewe ExcessNone | 100 | NCIH460 | ATP Lite | 72h |

71

84

94

95 112

48

69

77

85

93 116

46

47

69

86

.0012 .0037

99

.011 .033

11

-8

-5

-1

.1

Eribulin(E7389) (uM)

.0012 .0037

.011 .033

Trametinib (uM)

65

93 111 128

10

12

38

41

-24 -19

19

.1

89 127 151 156 161 173


84 112 141 145 158 171

48 109 131 133 147 162


0

Eribulin(E7389) (uM)

89 100 101 118 149


.0012 .0037

.011 .033

.0012 .0037

.011 .033

33

30

73

73

-1

14

28

48

61

-8

-7

.1

75

81

16

14

.0012 .0037

94 116 130
46

47

66

.011 .033

.1

.0012 .0037

.011 .033

Eribulin(E7389) (uM)

Loewe ExcessNone | 100 |


A2780 | ATP Lite | 72h |

Dose MatrixNone | 100 | Hep G2


| ATP Lite | 72h |

Loewe ExcessNone | 100 | Hep


G2 | ATP Lite | 72h |

44

59

41

34

33

-2

35

55

39

29

30

24

45

29

26

28

-1

27

41

24

24

29

35

37

17

14

19

20

.0012 .0037

-15 -15

.011 .033

.1

DT-1308810

157 167 174 174 178 188


139 147 160 158 173 181
89 110 114 142 134 158
56

65

77

87

98 131

46

57

57

65

67

98

16

37

50

77

Eribulin(E7389) (uM)

.0012 .0037

.011 .033

E6201

.1

Eribulin(E7389) (uM)

Eribulin(E7389) (uM)

27

65

DT-1308809

.1

62

99 122 128 125 168 168

Eribulin(E7389) (uM)

DT-1308809

Growth Inhibition (%) Vol=6.67(.45)


Chi2=53

85

75

N=2

.025 .074 .22

58

Trametinib (uM)

94 101 122 139

81

Growth Inhibition (%)

.66

61

.1

Dose MatrixNone | 100 | NCIH460 | ATP Lite | 72h |

DT-1308808

-8

Eribulin(E7389) (uM)

91 121 131 151 151

Trametinib (uM)

11

.0012 .0037

Eribulin(E7389) (uM)

66

Growth Inhibition (%) Vol=4.31(.43)


Chi2=15

N=1-2

ANDI(E6201) (uM)

Growth Inhibition (%)

99

.011 .033

Eribulin(E7389) (uM)

.025 .074 .22

ANDI(E6201) (uM)

37 119 136 139 165 169

50

Growth Inhibition (%) Vol=9.53(.86)


Chi2=40

.12

-4

39

DT-1308810

.1

15

22

22

26

36

13

27

24

40

47

-12

12

41

32

56

-6

13

20

24

50

15

23

19

16

22

-7

Eribulin(E7389) (uM)

.0012 .0037

.011 .033

Growth Inhibition (%) Vol=6.16(.49)


Chi2=17

86 101

27

10

64

22

N=2

44

16

3.3

33

1.1

-1

70 139 150 155 166 177

.37

27

61

.12

10

56

ANDI(E6201) (uM)

41

-2

41

Growth Inhibition (%)

42

-3

121 165 164 179 185 186

9.1e-4 .0027 .0082

.37

56

1e-4 3e-4

97 121

50

79

47

N=1-2

58

38

Trametinib (uM)

55

-13 35

DT-1308818

Growth Inhibition (%)

19

94 162 165 174 177 184

27

37

10

22

21

37

3.3

12

22

34

1.1

15

21

30

.37

21

21

-3

143 177 181 183 184 188

.12

16

50

ANDI(E6201) (uM)

1.1

50

98 117

48

9.1e-4 .0027 .0082

89

46

1e-4 3e-4

87

46

Trametinib (uM)

76

-3

Loewe Excess | None | 100 |


Hep G2 | ATP Lite | 72h

164 183 183 184 184 190

1.1

55

138 187 187 189 191 192

Growth Inhibition (%) Vol=8.91(.19)


Chi2=580

38

Dose Matrix | None | 100 | Hep


G2 | ATP Lite | 72h

DT-1308818

Growth Inhibition (%) Vol=8.17(.48)


Chi2=67

42

10

37

34

3.3

21

35

1.1

31

.37

3.3

-4

32

.12

90 111 128 132

30

74

11

.025 .074 .22

3.3

63

171 190 192 191 195 194

.0027 .0082

50

42

N=2

34

ANDI(E6201) (uM)

36

DT-1308813

Growth Inhibition (%)

14

Loewe Excess | None | 100 |


A2780 | ATP Lite | 72h

DT-1308813

N=2

10

Hep G2

Dose Matrix | None | 100 |


A2780 | ATP Lite | 72h

DT-1308817

Trametinib (uM)

82 105 108 128 144

DT-1308817

Growth Inhibition (%)

10

71

.37

A2780

Loewe Excess | None | 100 |


NCI-H460 | ATP Lite | 72h

.12

NCI-H460
Dose Matrix | None | 100 | NCIH460 | ATP Lite | 72h

Trametinib

.1

Eribulin(E7389) (uM)

Figure 5: Eribulin x MEK inhibitor combination activities. Select dose matrices (Growth Inhibition and Loewe Excess) for eribulin in combination with the MEK
inhibitors E6201 and trametinib are shown for cell lines NCI-H460, A2780 and Hep G2. Breadth of combination activity is shown in Figure 2 (Synergy Score heat
map).

KATOIII

MDA-MB-231

T47D

NCI-H460

Hep G2

786-0

T24

A375

9.21

12.9

5.24

13.6

13

12.2

14.9

8.76

8.53

22

15.1

6.99

13.1

9.49

14.5

11.8

.0012 .0037

.011 .033

.1

Eribulin(E7389) (uM)

42

68

.0012 .0037

74

75

.011 .033

-21 -11

14

.0012 .0037

.011 .033

.1

Eribulin(E7389) (uM)

36 100 140 146 149

17

85 128 138 148

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Eribulin(E7389) (uM)

-1

39

-5

35

12

15

-3

-4

-5

.011 .033

10

Chi2=180

.1

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Eribulin(E7389) (uM)

41

63

78

86 160 166

27

72

81 129 150

-6

24

54

82 103 153

12

13

42

76 104 125

19

11

64

78

94 125

42

65

91 101

1.4e-4 4.1e-4 .0012 .0037

.011

Eribulin(E7389) (uM)

Loewe Excess | None | 100 |


SNG-M | ATP Lite | 72h

21

36

18

69

66

-8

31

13

38

50

-6

13

14

12

52

12

-5

13

24

19

-4

25

10

24

-5

-3

1.4e-4 4.1e-4 .0012 .0037

Chi2=8

13

.0012 .0037

Growth Inhibition (%) Vol=5.24(.53)

27

Eribulin(E7389) (uM)

.31

16

31

.31

-9

.019 .039 .078 .16

-3

-7

-1

31 119 138 147 148

-9

ABT-263 (uM)

12

.1

Dose Matrix | None | 100 | SNGM | ATP Lite | 72h

Growth Inhibition (%) Vol=2.33(.36)


Chi2=13

35

-9

10

28

16

37

2.5

13

27

1.2

55 123 141 147 151

.62

21

113 116 113 120 118

SNG-M

39

N=1-2

28

22 108 115 115 116 115

Eribulin(E7389) (uM)

Loewe Excess | None | 500 | SK


-BR-3 | ATP Lite | 72h

ABT-263 (uM)

20

58 115 138 147 150

76

N=2

10

-18 98 100 101 99 102

Growth Inhibition (%) Vol=22(.36)

-1

-4

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

69

2.5

53 168 187 190 192 190

45

69

1.2

-7

62 184 190 189 196 194

41

65

.62

99

43

67

ABT-263 (uM)

98

25

36

Growth Inhibition (%)

69 102 101

28

Eribulin(E7389) (uM)

Growth Inhibition (%)

18

10

17

-1

42

2.5

34

1.2

34

10

N=2

31

.62

94 108

28

83

-11 19

ABT-263 (uM)

73

37

Chi2=29

68

35

Growth Inhibition (%) Vol=5.56(.3)

29

99 110 121

13

41

.019 .039 .078 .16

76 103 122 139 134 135

15

2.5

91

17

40

-32 65

ABT-263 (uM)

1.2

83

3
26

74 190 192 193 191 194

SK-BR-3

.62

25

58 100 103

95 192 193 192 196 196

Dose Matrix | None | 500 | SKBR-3 | ATP Lite | 72h

29

ABT-263 (uM)

94 107 110 122 129

95

Loewe Excess | None | 500 |


MCF7 | ATP Lite | 72h

1.2

37

Growth Inhibition (%)

10
5
2.5

57 109 112 114 122 133

.62

ABT-263 (uM)

69 113 122 125 128 128

102 124 110 125 131 132

91

15

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

MCF7
Dose Matrix | None | 500 |
MCF7 | ATP Lite | 72h

73

-3

41

98

2.5

Eribulin(E7389) (uM)

98

Loewe Excess | None | 100 |


MDA-MB-231 | ATP Lite | 72h

N=2

-1
.011

Eribulin(E7389) (uM)

-25 -36 -13 73

178 194 195 197 195 199

1.2

74 117 190 194

71

.62

1.4e-4 4.1e-4 .0012 .0037

67 151 194 195

22

71

ABT-263 (uM)

-3

46

29

69

.011

15

10

86

89

90

48

2.5

96

58

39

1.2

74

35

85 146 194 198

13

.62

27

32

52

34

47

N=2

-5
17

80 167 193 193

ABT-263 (uM)

95

53

MDA-MB-231
Dose Matrix | None | 100 | MDA
-MB-231 | ATP Lite | 72h

Loewe Excess | None | 100 |


KYSE-410 | ATP Lite | 72h

Growth Inhibition (%)

92

10

70

14

60

2.5

20

ABT-263 (uM)

11

10

N=2

64

1.2

84

58

140 165 175 196 198 197

.62

82

55

Eribulin(E7389) (uM)

12

61

14

Dose Matrix | None | 100 | KYSE


-410 | ATP Lite | 72h

Growth Inhibition (%) Vol=11.8(.72)


Chi2=110

24

1.4e-4 4.1e-4 .0012 .0037

12

-23 -13 13

2.5

75 125 171 171

16

1.2

79 146 179 174

50

24

.62

66

22

20

ABT-263 (uM)

15

16

90 152 177 181

Growth Inhibition (%)

10
2.5

85

70

1.2

100 109 135 177 180 187

.62

ABT-263 (uM)

180 180 183 187 191 183

KYSE-410

Loewe Excess | None | 100 |


NCI-H1650 | ATP Lite | 72h

Growth Inhibition (%) Vol=13.6(.49)


Chi2=1200

NCI-H1650
Dose Matrix | None | 100 | NCIH1650 | ATP Lite | 72h

Growth Inhibition (%)

0.928 0.663

HT-1080

2.59

PC-3

11.8

MIA PaCa-2

NCI-H522

12.6

FaDu

NCI-H1650

2.33

KYSE-410

A549

12.1

SNG-M

SK-BR-3

5.77

SK-OV-3

MDA-MB-468

5.56

Eribulin-Insensitve Cell Lines

A2780

MDA-MB-436

ABT-263

NCI-H69

Eribulin +

BCL2

NCI-H526

Target

MCF7

Eribulin-Sensitive Cell Lines

.011

Eribulin(E7389) (uM)

Figure 6: Synergy Score heat map and representative Growth Inhibition dose matrices for eribulin x ABT-263. Select pairs of dose matrix plots for eribulin
in combination with the BCL-2 family inhibitor ABT-263 are shown for cell lines NCI-H1650, KYSE-410, MDA-MB-231, MCF7, SK-BR-3 and SNG-M. Three strong
synergies are highlighted (black arrows, top row of matrices) as well as cell lines with weak combination effects (grey arrows, bottom row of matrices).

preclinical cancer models [4,7] and numerous monotherapy


clinical trials are ongoing to investigate efficacy in non-breast
tumor types (see www.clinicaltrials.gov). Consistent with utility
for additional potential indications, a variety of tumor cell lines
including lung, ovarian, endometrial, head and neck, prostate and
melanoma are shown here to be sensitive to eribulin at clinically
relevant concentrations. Since tumor cells have a robust capacity to
lessen the therapeutic effects of cancer drugs through adaptive and
acquired resistance mechanisms, the identification of drugs that can

014

paired with eribulin to trigger selective and synergistic tumor cell


death represents a critical path toward optimizing patient benefit.
Specifically, drug combinations need to be identified for situations
in which the effects of eribulin monotherapy are suboptimal, or in
which intrinsic or acquired drug resistance might be overcome with
strategically selected drug combinations.
Not only eribulin, but also all anticancer drugs need to be looked
for appropriate combination partners. A molecular targeted agent,

Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identification of Combinatorial Drugs that Synergistically Kill both EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.

Rickles, et al. (2015)

Eribulin-Insensitve Cell Lines

2.01

-0.56

1.18

1.83

0.94

-1.44

1.47

1.10

0.09

-0.93

-1.52

-1.94

3.05

3.44

1.94

2.11

0.74

1.05

1.22

-2.97

-8.20

-0.48

-4.03

-5.42

-2.15

-2.43

-6.28

1.09

-2.28

-5.81

-5.53

-10.65

-2.28

-7.96

0.32

-0.05

-0.69

0.35

-1.21

Topotecan

-1.48

-0.59

-3.14

-4.16

-0.17

-1.87

-5.33

-1.87

-3.47

-4.55

-1.79

-3.49

-5.15

1.19

-1.76

-1.68

-4.50

-11.00

-0.39

-2.89

-0.17

1.14

0.31

-0.88

-0.68

Gemcitabine

-4.28

-3.41

-4.49

-4.48

-0.02

-0.88

-5.23

-0.11

-1.72

-2.30

-0.24

-1.85

-3.07

0.65

-2.00

-2.19

-2.99

-13.86

-0.73

-8.03

-0.23

0.86

1.09

-1.43

-2.55

-7

11

25

62

89 108

.37

23

17

44

73

82

70

-9

29

56 113 104

.12

32

47

86 108 122

43

74 115 120

53

64 106 136 150

40

92 144 152 155

Eribulin(E7389) (uM)

82

80

61

82

84

.011

Eribulin(E7389) (uM)

92

90 102 93 106 118

73

67

66

82

56

51

90 100 100

20

63 121 130 130

53
0

91

1.4e-4 4.1e-4 .0012 .0037

98

79

87

99

20

42

75 110 156

-8

35

82 139 180

22

57

96 151 180

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

18

10

13

12

-4

-10

-3

-29 -28

-8

-22 -11

-44

20

20

25

-32

29

36

46

53

35

47

52

56

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Eribulin(E7389) (uM)

1.4e-4 4.1e-4 .0012 .0037

63
.011

KYSE-410

HT-1080

Dose MatrixNone | 100 | KYSE410 | ATP Lite | 72h |

Dose MatrixNone | 100 | HT1080 | ATP Lite | 72h |

96

96 102 97 104 118

118 111 109 111 108 120

92

97

74

93 101 112 103 113

26

91 111 121 126 145

.011

Eribulin(E7389) (uM)

7
0

97 105 102 107

89 110 117 125 152


89 108 115 133 159
.0012 .0037

.011 .033

.1

Eribulin(E7389) (uM)

1.1

DT-1308945

76

73

72

70

75

78

48

39

46

44

55

50

25

26

28

40

37

53

-5

27

64

89

92

-5

43

79

87

99

5
0

Cytarabine

Eribulin(E7389) (uM)

Dose MatrixNone | 100 | A2780


| ATP Lite | 72h |

DT-1308942

T24

786-0

Hep G2

T47D

KATOIII

PC-3

FaDu

SNG-M

43

9.9

N=2

-5

14

Eribulin(E7389) (uM)

N=2

N=1-2

N=2-3

65

87 116

.37

51

24

1.4e-4 4.1e-4 .0012 .0037

54

76

.014 .041 .12

37

12

31

63

16

23

15

49

Topotecan Hydrochloride
(uM)

63

47

Growth Inhibition (%)

59

41

30

71

34

59

Topotecan Hydrochloride
(uM)

76

27

.014 .041 .12

67

30

93 111 139

64 106 136 150

54

28

Topotecan Hydrochloride
(uM)

68

53

54

Growth Inhibition (%)

65

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

.014 .041 .12

82

73

42

DT-1308947

DT-1308943

Topotecan Hydrochloride
(uM)

69

Growth Inhibition (%)

1.1
.37
.014 .041 .12

31

71

49

52

DT-1308940

13
-16

.014 .041 .12

.37

123 119 122 136 127 136

63

43

95 111

.0015 .0046

1.1

109 103 124 115 111 117

53

35

91

A2780

146 143 141 149 148 154

113 130 109 113 113 105

44

NCI-H69
Dose MatrixNone | 500 | NCIH69 | ATP Lite | 72h |

1.1

NCI-H1650
Dose MatrixNone | 100 | NCIH1650 | ATP Lite | 72h |

160 160 156 164 167 172

50

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

162 154 157 153 154 158

DT-1308946

50

Eribulin(E7389) (uM)

.37

MDA-MB-468

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Eribulin(E7389) (uM)

3.3

95 119 129 131

1.1

50

44

.37

35

44

.12

98 107 109

44

90

40

Cytarabine (uM)

80

Growth Inhibition (%)

1.1
.37
.014 .041 .12

Cytarabine (uM)

74

N=2-3

99 111 111

Dose MatrixNone | 100 | MDAMB-468 | ATP Lite | 72h |

176 177 181 183 183 179

98

N=2

Growth Inhibition (%)

9.9

Cytarabine (uM)

N=2

.011

91

Growth Inhibition (%)

1.4e-4 4.1e-4 .0012 .0037

Growth Inhibition (%)

1.1

Cytarabine (uM)

90

90

N=2

62

81

Growth Inhibition (%)

52

78

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Eribulin(E7389) (uM)

DT-1308944

190 186 191 190 194 193


144 152 158 166 156 168
115 117 118 125 129 145
88
59
0

88

91

92

95 108

71

74

78

84

22

57

96 151 180

90

N=2

60

101 111 111 116 128 136

Growth Inhibition (%)

50

44

9.9

53

54

55

3.3

1.1

34

56

53

1.1

63

56

52

.37

63

44

46

.12

42

55

39

Cytarabine (uM)

28

51

3.3

24

89 102 98 106 113

9.9

.37
.014 .041 .12

13

96

3.3

98 101 94 105 119 125

78

1.1

91

64

.37

89

78

.12

86

58

77

62

N=2

86

47

Growth Inhibition (%)

69

63

N=2

64

63

Topotecan Hydrochloride
(uM)

68

64

Growth Inhibition (%)

72

41

1.1

69

60

KATOIII
Dose Matrix | None | 500 |
KATOIII | ATP Lite | 72h

DT-1308937

.37

59

36

HT-1080
Dose Matrix | None | 100 | HT1080 | ATP Lite | 72h

DT-1308939

.014 .041 .12

69

Eribulin(E7389) (uM)

KYSE-410
Dose Matrix | None | 100 | KYSE
-410 | ATP Lite | 72h

DT-1308938

Cytarabine (uM)

Dose Matrix | None | 500 | SKBR-3 | ATP Lite | 72h

DT-1308941

N=1-2

SK-BR-3

Dose Matrix | None | 100 | MDA


-MB-468 | ATP Lite | 72h

Growth Inhibition (%)

MDA-MB-468

DT-1308936

Topotecan Hydrochloride
(uM)

A2780

MDA-MB-436
Dose Matrix | None | 100 | MDA
-MB-436 | ATP Lite | 72h

A375

0.84

-1.32

NCI-H460

NCI-H526

-2.30

-5.94

HT-1080

NCI-H522

-5.95

-8.73

MIA PaCa-2

A549

-5.07

-5.39

KYSE-410

SK-BR-3

0.10

-3.92

SK-OV-3

MDA-MB-468

-3.35

Cytarabine

NCI-H69

MDA-MB-436

17-DMAG

NCI-H1650

Eribulin +

MCF7

MDA-MB-231

Eribulin-Sensitive Cell Lines

Topotecan

4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Eribulin(E7389) (uM)

Figure 7: Loewe Volume heat map of select compounds screened in combination with eribulin. Loewe Volume scores are shown with potential synergies in
pink/red and potential antagonisms in aqua/blue. Also shown are Growth Inhibition dose matrices for eribulin x cytarabine and eribulin x topotecan in the cell lines
MDA-MB-436, MDA-MB-468, SK-BR-3, KYSE-410, HT-1080 and Kato III.

vemurafenib (BRAF inhibitor), demonstrated 48% anti-tumor


response and extended PFS to 5.3 months in V600E melanoma
patients in the BRIM3 study [21], in a first-line BRAF mutated
melanoma therapy. However, the treatment of vemurafenib caused
resistance in some patients through its 2-18 months post treatment
[22]. Recently, four articles reported five resistance mechanisms
for vemurafenib [23-27]. Those authors attributed their resistance
mechanisms to the overexpression of PDGFR- tyrosine kinase [23],
and/or Q61K NRAS [24], COT kinase [25], IGF-1R [26], C121S
MEK [27], in melanoma cells. Selective inhibitors against those
molecules may be appropriate combination partners to prohibit these
resistant mechanisms in V600E melanoma patients. Comprehensive
combination analysis by using comprehensive compounds on several
cancer cells harboring different molecular mutation are very useful to
find out appropriate combination partners.
In this study, we describe the identification of approved and
emerging drugs that synergize when combined with eribulin, with
good breadth of combination activity observed in the twenty five cell
line panel used for screening. Synergy is observed in both eribulinsensitive and insensitive cell lines, suggesting potential clinical benefit
for both responders and non-responders of eribulin monotherapy.
In addition to approved drugs that target EGFR/HER2 (erlotinib,
lapatinib, BIBW2992/afatinib), mTOR (everolimus), and MEK
(trametinib), we show that emerging drugs such as BEZ235 (panPI3K/mTOR), BKM-120 (PI3K), and ABT-263 (BCL-2 family
inhibitor) strongly synergize with eribulin in certain cell lines.
Furthermore, the use of both target-specific (EGFR/HER2, MEK)
and pathway-specific (PI3K), mechanistically redundant compounds

015

in our studies validates the identified target specificities as being


important for synergy. By evaluating a wide concentration range
for these molecules, we can interrogate combination activities and
evaluate target selectivity; in addition, where pharmacokinetic data
are available, we can obtain initial insights as to whether such effects
could occur at clinically relevant concentrations. Looking at the
eribulins concentrations which showed synergy effects combined
with these approval drugs, we found that they seem to be relatively
wide-range. Not only at the highest concentration but also at the
lower concentrations, eribulin could synergize with several drugs.
One challenge with the evaluation of in vitro drug activities is that
patterns of drug exposure may not match what can be achieved in
vivo. Limited drug exposure (pulse dosing) could be used to compare
in vitro results with exposure in a clinical setting. In addition,
tumor bearing animals could be treated with both drugs to validate
combination activities to examine combination drug toxicities. To
this end, in vivo animal studies are currently in progress; to date,
eribulin combination activities with erlotinib, everolimus, and BKM120 have been reproduced in human tumor xenograft models in
mice, with these combinations showing acceptable toxicity profiles
(manuscript in preparation).
An important goal for any monotherapy or drug combination is
the identification of predictors of response so that clinicians can select
the patient population most likely to benefit from treatment. The
screen described here represents the discovery phase of such a project
and additional work is required to identify predictors of response with
a certainty that can drive patient selection. For instance, the kinetics

Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identification of Combinatorial Drugs that Synergistically Kill both EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.

Rickles, et al. (2015)

of the appearance of combination activity should be explored, and the


relationships, if any, of synergy/non-synergy to intrinsic or acquired
resistance should be further defined. To power such analyses,
additional cell lines could be screened to generate a larger data set
for more detailed genetic characterization. Additional analyses using
larger data sets with sufficient breadth of known genetic profiles will
be required to generate a fully vetted understanding of response
prediction.

3. Smith JA, Wilson L, Azarenko O, Zhu X, Lewis BM, et al. (2010) Eribulin binds
at microtubule ends to a single site on tubulin to suppress dynamic instability.
Biochemistry 49:13311337.

In addition to identifying drug synergies, we have also identified


antagonistic combinations. For example, low concentrations of
cytarabine or topotecan have little effect on proliferation as single
agents, but can dramatically antagonize eribulin activity under
combination conditions. Indeed, this is not without precedent:
antagonism has been observed for a variety of cancer drug
combinations in preclinical studies, prompting follow up analyses
to investigate both the mechanisms of the synergies as well as the
effects of drug combination sequencing [28-35]. For instance, in
one extensively studied example, the anthracycline doxorubicin
antagonized activity of the vinca alkaloid vincristine in 83% (15/18)
of hematopoietic cell lines tested. This antagonism was reproduced
in vivo in a xenograft model and was also observed in 34% (12/35) of
childhood leukemia cells simultaneously treated with both drugs ex
vivo [35]. Moreover, combination activity was shown to be sequence
dependent: if cells are exposed to the anthracycline first, antagonism
is observed, but if exposure to vinca alkaloid precedes anthracycline,
the combination result is beneficial. A p53-dependent mechanism for
this antagonism was proposed, where anthracycline effects stabilize
anti-apoptotic BCL2 family members, thus reducing cytotoxic effects
of the vinca alkaloid. With respect to our current study, the eribulin
antagonisms observed with 17-DMAG, cytarabine, topotecan and
gemcitabine do not necessarily require that p53 be functional as was
the case in the preceding example, but further study will be required to
determine this. Indeed, the mechanisms by which these compounds
antagonize eribulin may overlap or be entirely unrelated.

6. Swami U, Chaudhary I, Ghalib MH, Goel S. (2012) Eribulin--A review of


preclinical and clinical studies. Crit Rev Oncol Hemat 81:163184.

In conclusion, we have identified promising preclinical in


vitro synergy combination partners with eribulin, with compound
mechanistic redundancy helping to validate particular targets and
pathways as important for selective, synergistic tumor cell killing.
In order to utilize the current results with the goal of clinical
implementation, further studies, including sequencing of drugs
and both in vivo efficacy and toxicology studies will be needed. Our
results suggest that further investigation is merited to assess whether
additional patient benefit with eribulin, in some patient populations,
may be achieved when eribulin is administered in the clinic as part of
a combination drug regimen in patients in the context of controlled
clinical trials.

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Copyright: 2015 Rickles RJ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

017

Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identification of Combinatorial Drugs that Synergistically Kill both EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.