Identification of Combinatorial Drugs that Synergistically Kill both

Global Journal of Cancer Therapy

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

009

eertechz

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 eribulin’s anticancer activity. Further elucidation of the mechanisms responsible for such interactions may be important for identifying valuable therapeutic partners for eribulin.

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 anti-cancer 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 MethodsMaterials

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

IntroductionEribulin 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 Phase 3 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

Research Article

Identification of Combinatorial Drugs that Synergistically Kill both Eribulin-Sensitive and Eribulin-Insensitive Tumor Cells

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*1Horizon Discovery Inc., MA, Japan2Eisai Inc., MA, Japan3Eisai Co., Ltd., Japan

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


Rickles, et al. (2015)

010

Toronto Research Chemicals. Cell proliferation was measured by ATP Lite 1step Luminescence Assay System (Perkin Elmer).

MethodsCells were thawed and grown in culture media according to

vendor’s 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 Horizon’s proprietary ChaliceTM Analyzer software.

Combination High-Throughput ScreeningCells 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 Horizon’s 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:

00

0

00

0

If : :

If

100* (1 )

100* ( : : 1 )

T VVTV

TT VVV V

−< −

−≥ −

−

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 resultsTo 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

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 self-crosses 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 (2σ’s) or three standard deviations (3 σ’s) can be considered candidate synergies at the 95% and 99% confidence levels, respectively.

ResultsEvaluation of eribulin potency using a cell-based assay

Eribulin’s 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



011

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 2σ’s 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 3σ 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.

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.

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, BIBW-2992 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 inhibitorsDysregulation 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

MC

F7M

DA-

MB

-231

MD

A-M

B-4

36M

DA-

MB

-468

SK-B

R-3

T47D

A549

NC

I-H16

50N

CI-H

460

NC

I-H52

2N

CI-H

526

NC

I-H69

A278

0SK

-OV-

3SN

G-M

Hep

G2

KYS

E-41

0Fa

Du

786-

0T2

4M

IA P

aCa-

2PC

-3A3

75H

T-10

80K

ATO

III

0.00.10.20.30.40.50.60.70.80.91.0

5.0015.0025.0035.00

Erib

ulin

GI 5

0 (n

M)

Bre

ast

Lung

Ova

rian

Endo

met

rial

Live

rEs

opha

geal

Hea

d+N

eck

Kid

ney

Bla

dder

Pan

crea

sPr

osta

teM

elan

oma

Fibr

osar

com

aSt

omac

h

Hep

G2

T24

MD

A-M

B-2

31N

CI-H

460

786-

0T4

7DA3

75A2

780

KAT

OIII

FaD

uN

CI-H

1650

A549

MC

F7N

CI-H

526

SNG

-MN

CI-H

522

NC

I-H69

MD

A-M

B-4

36K

YSE-

410

PC-3

MIA

PaC

a-2

HT-

1080

SK-B

R-3

SK-O

V-3

MD

A-M

B-4

68

0.00.10.20.30.40.50.60.70.80.91.0

5.0015.0025.0035.00

Erib

ulin

GI 5

0 (n

M)

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.

http://www.peertechz.com/Cancer-Therapy/pdf/GJCT-1-s104.rar







012

Eribulin-Sensitive Cell Lines Eribulin-Insensitve Cell Lines

Target Eribulin + MCF

7

MDA

-MB-

436

MDA

-MB-

468

SK-B

R-3

A549

NCI-H

1650

NCI-H

522

NCI-H

526

NCI-H

69

A278

0

SK-O

V-3

SNG-

M

KYSE

-410

FaDu

MIA

PaCa

-2

PC-3

HT-1

080

KATO

III

MDA

-MB-

231

T47D

NCI-H

460

HepG

2

786-

0

T24

A375

ErbB1/ErbB2

BIBW-2992 1.31 1.80 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

Erlotinib 3.14 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

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 11.77

PI3K Pathway

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

MEKTrametinib 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.

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

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

N=2

DT-1308660

-

73

82

90

90

109

-1

74

93

98

109

149

53

94

135

149

170

183

85

130

160

168

165

185

88

129

142

164

167

184

102

146

174

175

174

187

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

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

Vol=

13.1

(.41)

Chi2=

270

DT-1308660

-

3

-15

-6

-6

12

-6

-15

-3

2

12

53

2

-1

38

52

74

86

-6

35

63

72

68

88

-8

33

46

67

71

88

6

51

78

79

78

91

Dose MatrixNone | 100 | 786-0 | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=2

DT-1308661

-

-2

0

1

10

91

27

25

36

60

78

167

32

44

78

97

89

173

42

76

102

118

113

189

71

94

113

129

134

191

112

126

129

113

117

197

Loewe ExcessNone | 100 | 786-0 | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

Vol=

11.1

(.35)

Chi2=

190

DT-1308661

-

-2

0

1

0

0

15

13

23

44

49

74

5

17

51

65

44

78

-10

24

48

61

42

91

-10

12

31

45

45

89

9

23

27

9

13

92

Dose MatrixNone | 100 | T24 | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=2

DT-1308662

-

-7

-4

9

1

61

-6

-6

1

44

73

98

-4

-1

40

83

107

105

13

54

79

101

127

127

63

76

94

124

135

140

91

111

109

126

126

135

Loewe ExcessNone | 100 | T24 | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

Vol=

11.3

(.55)

Chi2=

150

DT-1308662

-

-7

-4

9

0

0

-7

-6

1

44

69

37

-5

-2

39

81

96

41

2

42

65

84

92

59

-1

11

30

57

65

70

1

21

19

36

36

45

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

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

N=2

DT-1308663

-

-3

7

6

22

66

20

38

41

63

72

86

30

52

77

87

90

97

53

75

96

110

105

135

74

96

106

122

121

142

96

119

115

123

118

166

Loewe ExcessNone | 100 | A375 | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

Vol=

9.22

(.44)

Chi2=

80

DT-1308663

-

-3

7

5

-1

1

6

23

25

43

33

19

-1

20

43

50

38

29

-2

19

39

51

40

66

-4

18

29

44

43

64

5

27

23

31

26

74

Dose MatrixNone | 100 | NCI-H460 | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=2

DT-1308664

-

6

-4

-4

5

29

38

27

49

65

94

96

49

53

84

98

100

100

55

79

107

135

119

103

91

99

121

130

111

111

114

120

118

121

120

142

Loewe ExcessNone | 100 | NCI-H460 | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

Vol=

7.77

(.5)

Chi2=

270

DT-1308664

-

6

-4

-4

0

0

12

1

22

37

65

67

2

6

38

51

53

53

-17

8

36

64

48

31

-1

6

28

37

18

18

8

14

12

15

14

36

Dose MatrixNone | 100 | MDA-MB-468 | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

N=2

DT-1308665

-

10

31

34

56

120

54

65

80

86

91

173

90

102

113

128

139

187

150

140

156

154

172

192

164

169

167

170

179

194

165

165

174

174

181

196

Loewe ExcessNone | 100 | MDA-MB-468 | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

Vol=

4.7(

.48)

Chi2=

66

DT-1308665

-

6

17

-2

-19

11

6

13

22

16

-1

59

-10

0

11

23

25

64

8

-2

14

12

30

50

3

9

7

10

19

34

-2

-2

7

7

14

29

Dose MatrixNone | 100 | KYSE-410 | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

N=2

DT-1308666

-

24

48

62

74

181

-3

28

37

69

73

186

37

56

57

74

91

193

80

79

91

96

107

192

78

105

116

143

145

193

97

115

139

154

160

185

Loewe ExcessNone | 100 | KYSE-410 | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

Vol=

4.13

(.49)

Chi2=

99

DT-1308666

-

15

22

-1

-41

24

-7

10

1

-1

-42

29

2

11

-1

-9

-24

36

-1

-3

7

7

-8

34

-12

15

26

53

30

36

6

25

49

64

45

27

Dose MatrixNone | 100 | SNG-M | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

N=2

DT-1308667

-

5

20

6

36

62

7

32

14

42

57

74

50

66

63

64

86

90

80

75

83

113

135

137

94

126

136

148

145

152

131

147

150

149

146

151

Loewe ExcessNone | 100 | SNG-M | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

Vol=

5.87

(.48)

Chi2=

37

DT-1308667

-

4

17

-6

-1

2

-11

13

-9

13

11

12

9

23

18

16

29

24

5

-1

7

36

58

61

-14

19

29

41

38

44

4

20

22

22

19

24

Dose MatrixNone | 500 | MCF7 | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=2

DT-1308668

-

-4

19

24

46

132

69

68

75

73

88

156

76

75

77

71

92

161

81

82

77

82

114

181

93

89

100

102

127

188

115

120

114

118

151

194

Loewe ExcessNone | 500 | MCF7 | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

Vol=

2.46

(.26)

Chi2=

330

DT-1308668

-

-4

19

22

-3

4

6

-1

-7

-19

-9

27

-21

-22

-20

-26

-5

32

-16

-14

-20

-15

18

52

-3

-7

3

5

31

59

18

23

17

21

54

65

Dose MatrixNone | 100 | Hep G2 | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th I

nhib

ition

(%

)

N=3-

4

DT-1308669

-

5

-3

11

7

75

19

25

33

48

69

115

21

34

48

78

82

117

42

58

76

85

88

128

56

73

71

82

82

135

73

94

86

76

70

131

Loewe ExcessNone | 100 | Hep G2 | ATP Lite | 72h |

Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

Vol=

7.01

(.44)

Chi2=

20

DT-1308669

-

5

-3

11

0

0

7

13

20

33

43

39

-4

8

23

49

43

41

-1

15

31

39

34

53

-4

13

11

21

17

59

4

24

16

5

-2

54


Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=2

DT-1308670

-

13

3

25

30

86

90

93

92

93

98

154

108

115

121

133

127

174

123

132

110

130

122

176

144

136

139

138

136

180

153

156

160

158

159

191


Lapa

tinib

(uM

)0

.12

.37

1.1

3.3

9.9

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

Vol=

2.33

(.51)

Chi2=

6.1

DT-1308670

-

12

2

16

-13

8

5

6

-2

-1

8

65

-29

-22

-15

-4

-9

38

-14

-4

-27

-7

-15

40

8

-1

2

1

-1

43

16

19

23

21

22

54

MCF7 MDA-MB-468 NCI-H460

A2780 SNG-M Hep G2

KYSE-410 FaDu 786-0

T24 A375

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

(Figure 4). Combination activity is selective (for example, little or no combination activity in SK-BR-3, NCI-H552, NCI-H526, Mia PaCa-2, and 786-0) pointing to specific genetic determinants in responsive cell lines as being important for combination activity.

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



013



7

MD

A-M

B-43

6

MD

A-M

B-46

8

SK-B

R-3

A549

NCI

-H16

50

NCI

-H52

2

NCI

-H52

6

NCI

-H69

A278

0

SK-O

V-3

SNG

-M

KYSE

-410

FaD

u

MIA

PaC

a-2

PC-3

HT-

1080

KATO

III

MD

A-M

B-23

1

T47D

NCI

-H46

0

Hep

G2

786-

0

T24

A375

PI3K Pathway

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

AZD 8055

BEZ235

BKM-120

Everolimus

Dose Matrix | None | 100 | MDA-MB-468 | ATP Lite | 72h

BKM

-120

(uM

)0

.025

.074

.22

.66

2

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th I

nhib

ition

(%

)

N=2-

3

DT-1308717

-

7

-22

13

6

104

45

15

29

16

54

121

64

63

59

72

99

156

106

110

97

148

142

165

136

137

143

146

150

168

142

142

132

155

163

164

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

BKM

-120

(uM

)0

.025

.074

.22

.66

2

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=2

DT-1308718

-

13

13

19

32

94

63

63

57

79

92

129

90

98

108

99

106

127

101

106

113

123

127

141

98

103

107

104

120

128

72

74

67

83

104

132

Dose Matrix | None | 500 | NCI-H69 | ATP Lite | 72h

BKM

-120

(uM

)0

.025

.074

.22

.66

2

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

N=2

DT-1308719

-

1

16

34

74

164

27

20

41

58

93

170

62

62

76

98

140

171

126

136

133

148

160

171

143

135

151

158

167

175

147

153

156

160

168

174

Dose Matrix | None | 100 | A2780 | ATP Lite | 72h

BKM

-120

(uM

)0

.025

.074

.22

.66

2

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=2

DT-1308720

-

2

21

30

75

122

90

91

94

109

125

161

100

106

114

130

148

169

109

110

123

132

147

181

129

130

138

151

160

177

143

149

159

166

179

185

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

BKM

-120

(uM

)0

.025

.074

.22

.66

2

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th I

nhib

ition

(%

)

N=1-

2

DT-1308721

-

-3

18

9

23

84

6

44

37

43

66

106

54

60

62

75

84

142

89

81

84

86

95

141

92

91

99

100

113

158

99

111

114

128

162

164

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

BKM

-120

(uM

)0

.025

.074

.22

.66

2

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

N=2

DT-1308722

-

-5

12

8

19

85

25

26

30

35

59

99

56

63

66

70

77

145

89

92

92

96

119

161

124

145

150

141

174

171

163

175

181

178

180

186


BEZ2

35 (u

M)

0.0

12.0

37.1

1.3

31

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th I

nhib

ition

(%

)

N=1-

2

DT-1308723

-

24

49

67

81

100

-4

33

73

72

102

123

52

66

105

98

125

159

84

105

102

140

152

170

91

97

142

160

170

178

104

153

172

161

176

170


BEZ2

35 (u

M)

0.0

014

.004

1.0

12.0

37.1

1

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th I

nhib

ition

(%

)

N=1-

2

DT-1308724

-

25

22

48

93

103

61

33

66

103

127

153

82

88

105

149

173

173

120

134

146

173

184

185

143

168

173

182

191

186

158

165

176

182

187

186


BEZ2

35 (u

M)

0.0

12.0

37.1

1.3

31

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th I

nhib

ition

(%

)

N=1-

2

DT-1308725

-

55

97

107

104

100

60

83

103

120

123

125

99

100

125

134

141

139

102

115

140

150

146

138

90

118

142

156

150

145

67

95

132

145

141

147


BEZ2

35 (u

M)

0.0

014

.004

1.0

12.0

37.1

1

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

N=2

DT-1308726

-

16

13

57

103

139

15

33

32

52

113

148

58

83

85

112

148

165

126

140

147

155

164

163

144

147

158

166

169

170

146

159

163

165

174

180


BEZ2

35 (u

M)

0.0

014

.004

1.0

12.0

37.1

1

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=2

DT-1308727

-

33

58

89

99

103

89

94

105

109

138

155

108

134

131

143

159

164

115

127

134

147

163

167

133

139

156

166

173

176

159

165

168

180

180

175


BEZ2

35 (u

M)

0.0

014

.004

1.0

12.0

37.1

1

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

N=2

DT-1308728

-

18

30

77

82

95

22

40

62

99

115

128

57

72

85

104

126

130

96

105

112

153

163

167

151

157

173

175

179

175

180

181

187

184

188

188


AZD

8055

(uM

)0

.008

6.0

26.0

78.2

3.7

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=2

DT-1308693

-

54

76

90

97

103

91

110

110

126

137

152

104

138

134

150

158

166

108

143

152

157

167

170

128

152

170

173

177

177

147

177

179

182

184

178


AZD

8055

(uM

)0

.008

6.0

26.0

78.2

3.7

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

N=2

DT-1308706

-

11

32

57

77

87

14

38

52

70

91

117

64

74

83

92

110

142

86

102

109

140

147

155

135

146

171

174

176

179

174

179

184

187

185

184


AZD

8055

(uM

)0

.008

6.0

26.0

78.2

3.7

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th I

nhib

ition

(%

)

N=1-

2

DT-1308703

-

1

10

25

56

73

3

25

15

39

66

73

54

56

58

74

94

106

89

80

94

103

106

146

95

94

97

130

145

144

102

134

129

166

162

178


AZD

8055

(uM

)0

9.6e

-4.0

029

.008

6.0

26.0

78

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th I

nhib

ition

(%

)

N=2-

3

DT-1308694

-

3

14

17

26

47

52

38

26

55

62

61

64

69

60

71

100

121

106

107

109

132

157

174

136

135

152

159

180

184

150

154

142

162

174

179


AZD

8055

(uM

)0

.008

6.0

26.0

78.2

3.7

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

N=2

DT-1308699

-

19

39

57

104

132

21

46

62

84

104

132

61

87

100

123

143

149

130

147

153

159

163

159

140

161

163

165

162

164

150

162

164

168

166

169


AZD

8055

(uM

)0

.008

6.0

26.0

78.2

3.7

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=2

DT-1308695

-

35

56

81

99

100

71

76

80

104

109

128

100

96

103

116

130

126

100

109

112

113

132

141

94

106

106

128

129

141

66

79

92

111

125

127

MDA-MB-468 NCI-H69 A2780 FaDu HT-1080 T47D

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

Ever

olim

us (u

M)

06.

2e-4

.001

9.0

055

.017

.05

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

N=2

DT-1308895

-

33

37

34

42

44

-1

35

57

43

59

58

53

77

68

72

75

73

85

92

89

89

95

101

88

93

103

98

108

132

102

128

128

142

147

136

Dose MatrixNone | 100 | T47D | ATP Lite | 72h |

Ever

olim

us (u

M)

06.

2e-4

.001

9.0

055

.017

.05

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th I

nhib

ition

(%

)

N=1-

2

DT-1308897

-

29

36

15

31

32

51

83

75

74

74

84

88

112

108

106

105

106

104

111

117

120

111

129

94

104

114

118

118

106

73

90

90

81

96

102


Ever

olim

us (u

M)

07.

6e-6

2.3e

-56.

8e-5

2.1e

-46.

2e-4

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

N=2

DT-1308896

-

14

14

20

23

35

12

21

26

33

53

56

66

65

72

76

86

106

121

134

134

139

151

150

133

150

151

146

163

162

135

145

155

160

164

166


Ever

olim

us (u

M)

07.

6e-6

2.3e

-56.

8e-5

2.1e

-46.

2e-4

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

N=2

DT-1308898

-

7

2

25

39

64

54

81

72

84

117

119

90

91

127

130

162

158

150

154

157

163

176

185

164

171

172

185

185

187

165

168

177

186

183

187


Ever

olim

us (u

M)

07.

6e-6

2.3e

-56.

8e-5

2.1e

-46.

2e-4

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=2

DT-1308899

-

4

14

30

62

74

90

90

90

97

110

111

108

111

123

136

148

147

123

130

123

136

141

151

144

147

143

159

163

169

153

159

161

173

180

179

Dose MatrixNone | 100 | HT-1080 | ATP Lite | 72h |

Ever

olim

us (u

M)

06.

2e-4

.001

9.0

055

.017

.05

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

N=2

DT-1308900

-

53

50

54

48

51

43

74

70

73

82

76

54

96

104

97

112

107

122

153

145

144

144

147

150

171

173

169

177

175

168

182

184

180

185

175

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 InhibitionABT-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

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

Antagonistic drug pairingsAs 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.

DiscussionEribulin has broad anticancer activity in a wide variety of



014


AND

I(E6

201)

(uM

)0

.12

.37

1.1

3.3

10

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=

2

DT-1308813

-

37

70

94

138

171

91

119

139

162

187

190

104

136

150

165

187

192

108

139

155

174

189

191

128

165

166

177

191

195

147

169

177

184

192

194

Loewe Excess | None | 100 | A2780 | ATP Lite | 72h

AND

I(E6

201)

(uM

)0

.12

.37

1.1

3.3

10

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Gro

wth

Inh

ibiti

on (

%)

Vol

=8.

91(.

19)

Chi2

=58

0

DT-1308813

-

6

5

-13

-3

11

6

10

16

35

46

30

-24

9

22

38

46

32

-19

12

27

47

48

31

0

38

39

50

50

35

19

41

50

56

50

34


AND

I(E6

201)

(uM

)0

.12

.37

1.1

3.3

10

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th I

nhib

ition

(%

)

N=1-

2

DT-1308817

-

-1

19

55

63

71

46

33

55

76

74

82

47

44

58

87

90

105

69

64

79

89

111

108

86

86

97

98

128

128

99

101

121

117

132

144

Loewe Excess | None | 100 | NCI-H460 | ATP Lite | 72h

AND

I(E6

201)

(uM

)0

.12

.37

1.1

3.3

10

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Gro

wth

Inh

ibiti

on (

%)

Vol

=4.

31(.

43)

Chi2

=15

DT-1308817

-

-4

1

0

-4

2

11

-6

9

16

7

14

-8

-13

-2

21

21

36

-5

-10

5

15

37

34

-1

-1

10

12

42

42

5

7

27

22

38

50

Dose Matrix | None | 100 | Hep G2 | ATP Lite | 72h

AND

I(E6

201)

(uM

)0

.12

.37

1.1

3.3

10

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=

2

DT-1308818

-

65

99

121

143

164

16

75

122

165

177

183

14

81

128

164

181

183

46

94

125

179

183

184

47

116

168

185

184

184

66

130

168

186

188

190

Loewe Excess | None | 100 | Hep G2 | ATP Lite | 72h

AND

I(E6

201)

(uM

)0

.12

.37

1.1

3.3

10

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Gro

wth

Inh

ibiti

on (

%)

Vol

=9.

53(.

86)

Chi2

=40

DT-1308818

-

-1

4

-3

-3

2

6

8

27

41

30

21

-8

14

33

41

34

21

7

28

30

56

37

22

-7

48

73

61

37

21

2

61

73

62

42

27


Tram

etin

ib (

uM)

0.0

25.0

74.2

2.6

62

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=

2

DT-1308808

-

48

65

58

61

66

46

69

71

75

81

91

47

77

84

85

94

121

69

85

94

93

101

131

86

93

95

111

122

151

99

116

112

128

139

151

Loewe ExcessNone | 100 | NCI-H460 | ATP Lite | 72h |

Tram

etin

ib (

uM)

0.0

25.0

74.2

2.6

62

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Gro

wth

Inh

ibiti

on (

%)

Vol

=6.

67(.

45)

Chi2

=53

DT-1308808

-

0

2

-5

-2

3

11

7

8

12

18

27

-8

14

21

22

30

57

-5

11

20

20

27

57

-1

7

8

24

36

64

5

22

18

34

45

57


Tram

etin

ib (

uM)

0.0

027

.008

2.0

25.0

74.2

2

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=

2

DT-1308809

-

48

67

84

89

95

89

109

108

112

127

139

100

131

135

141

151

156

101

133

140

145

156

157

118

147

157

158

161

167

149

162

172

171

173

176


Tram

etin

ib (

uM)

0.0

027

.008

2.0

25.0

74.2

2Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Gro

wth

Inh

ibiti

on (

%)

Vol

=8.

17(.

48)

Chi2

=67

DT-1308809

-

0

-1

1

-2

0

20

35

27

24

35

44

6

37

41

45

55

59

-15

17

24

29

39

41

-15

14

24

26

29

34

6

19

29

28

30

33

Dose MatrixNone | 100 | Hep G2 | ATP Lite | 72h |

Tram

etin

ib (

uM)

01e

-43e

-49.

1e-4

.002

7.0

082

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th I

nhib

ition

(%

)

N=1-

2

DT-1308810

-

46

56

89

139

157

9

57

65

110

147

167

16

57

77

114

160

174

37

65

87

142

158

174

50

67

98

134

173

178

77

98

131

158

181

188

Loewe ExcessNone | 100 | Hep G2 | ATP Lite | 72h |

Tram

etin

ib (

uM)

01e

-43e

-49.

1e-4

.002

7.0

082

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Gro

wth

Inh

ibiti

on (

%)

Vol

=6.

16(.

49)

Chi2

=17

DT-1308810

-

15

-6

-12

6

5

3

23

4

9

13

15

0

19

13

12

27

22

3

16

20

41

24

22

-7

4

24

32

40

26

4

22

50

56

47

36

E6201

Trametinib

NCI-H460 A2780 Hep G2

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).

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

ABT-

263

(uM

)0

.62

1.2

2.5

510

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=2

-

25

37

57

69

102

68

83

94

109

113

124

73

91

107

112

122

110

83

99

110

114

125

125

94

110

122

122

128

131

108

121

129

133

128

132

Loewe Excess | None | 500 | MCF7 | ATP Lite | 72h

ABT-

263

(uM

)0

.62

1.2

2.5

510

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

Vol=

5.56

(.3)

C

hi2=

29

-

5

0

-1

-11

7

5

-1

5

17

19

29

-21

-3

13

18

28

15

-11

5

16

20

31

29

0

16

28

28

34

35

14

27

35

39

34

37

Dose Matrix | None | 500 | SK-BR-3 | ATP Lite | 72h

ABT-

263

(uM

)0

.62

1.2

2.5

510

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th I

nhib

ition

(%

)

N=1-

2

-

13

12

21

42

76

17

36

31

55

58

103

85

100

119

123

115

122

128

140

138

141

138

139

138

146

147

147

147

134

148

149

148

151

150

135

Loewe Excess | None | 500 | SK-BR-3 | ATP Lite | 72h

ABT-

263

(uM

)0

.62

1.2

2.5

510

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

Vol=

2.33

(.36)

Chi2=

13

-

12

6

-1

-7

6

-3

6

-5

3

-9

27

3

15

35

39

31

37

-4

8

6

9

6

7

-5

3

4

4

4

-9

4

5

4

6

6

-9


ABT-

263

(uM

)0

.62

1.2

2.5

510

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=2

-

53

62

74

95

178

42

168

184

190

192

194

68

187

190

192

193

195

74

190

189

193

192

197

75

192

196

191

196

195

76

190

194

194

196

199

Loewe Excess | None | 100 | MDA-MB-231 | ATP Lite | 72h

ABT-

263

(uM

)0

.62

1.2

2.5

510

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

Vol=

22(.3

6)

Ch

i2=18

0

-

22

5

-18

-32

25

0

108

113

98

65

40

0

115

116

100

67

41

0

115

113

101

65

43

0

116

120

99

69

41

0

115

118

102

69

45


ABT-

263

(uM

)0

.62

1.2

2.5

510

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

N=2

-

22

15

70

100

180

12

50

66

85

109

180

24

75

79

90

135

183

61

125

146

152

177

187

82

171

179

177

180

191

84

171

174

181

187

183

Loewe Excess | None | 100 | NCI-H1650 | ATP Lite | 72h

ABT-

263

(uM

)0

.62

1.2

2.5

510

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

Vol=

11.8

(.72)

Chi2=

110

-

17

-5

11

-23

14

5

32

34

20

-13

12

-3

35

27

14

13

16

1

58

74

70

55

20

3

90

96

92

58

24

-1

86

89

95

64

16

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

ABT-

263

(uM

)0

.62

1.2

2.5

510

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

N=2

-

29

15

47

60

140

1

22

46

52

53

165

41

74

67

85

80

175

73

117

151

146

167

196

91

190

194

194

193

198

95

194

195

198

193

197

Loewe Excess | None | 100 | KYSE-410 | ATP Lite | 72h

ABT-

263

(uM

)0

.62

1.2

2.5

510

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

Vol=

13.6

(.49)

Chi2=

1200

-

26

3

9

-25

13

-7

4

17

-3

-36

39

5

28

13

15

-13

48

-4

36

69

58

73

69

-1

98

102

100

98

71

1

99

101

103

98

71

Dose Matrix | None | 100 | SNG-M | ATP Lite | 72h

ABT-

263

(uM

)0

.019

.039

.078

.16

.31

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

N=2

-

19

12

-6

4

41

9

11

13

24

27

63

42

64

42

54

72

78

65

78

76

82

81

86

91

94

104

103

129

160

101

125

125

153

150

166

Loewe Excess | None | 100 | SNG-M | ATP Lite | 72h

ABT-

263

(uM

)0

.019

.039

.078

.16

.31

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

Vol=

5.24

(.53)

Chi2=

8

-

19

12

-6

0

0

-5

-4

-5

1

-8

21

5

25

1

13

31

36

-3

10

8

14

13

18

0

3

13

12

38

69

0

24

24

52

50

66



7

MD

A-M

B-43

6

MD

A-M

B-46

8

SK-B

R-3

A549

NCI

-H16

50

NCI

-H52

2

NCI

-H52

6

NCI

-H69

A278

0

SK-O

V-3

SNG

-M

KYSE

-410

FaD

u

MIA

PaC

a-2

PC-3

HT-

1080

KATO

III

MD

A-M

B-23

1

T47D

NCI

-H46

0

Hep

G2

786-

0

T24

A375

BCL2 ABT-263 5.56 5.77 12.1 2.33 12.6 11.8 2.59 0.928 0.663 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

NCI-H1650 KYSE-410 MDA-MB-231

MCF7 SK-BR-3 SNG-M

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

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,



015

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-β 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 eribulin-sensitive 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 (pan-PI3K/mTOR), BKM-120 (PI3Kα), 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

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 eribulin’s 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 BKM-120 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


Cyta

rabi

ne (u

M)

0.0

14.0

41.1

2.3

71.

1

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

N=2

DT-1308936

-

-9

-7

13

36

69

7

9

11

24

60

59

43

29

25

28

41

69

74

56

62

42

64

72

115

113

89

63

63

68

120

104

108

63

63

64


Cyta

rabi

ne (u

M)

0.0

14.0

41.1

2.3

71.

1

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

N=2

DT-1308937

-

-8

14

52

78

101

22

5

20

49

81

111

57

35

42

63

90

111

96

82

75

76

91

116

151

139

110

87

95

128

180

180

156

116

111

136

Dose Matrix | None | 500 | SK-BR-3 | ATP Lite | 72h

Cyta

rabi

ne (u

M)

0.0

14.0

41.1

2.3

71.

1

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

N=2

DT-1308938

-

35

74

90

96

98

40

50

80

91

89

101

92

95

90

98

102

94

144

119

98

99

98

105

152

129

107

111

106

119

155

131

109

111

113

125

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

Cyta

rabi

ne (u

M)

0.1

2.3

71.

13.

39.

9

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th I

nhib

ition

(%

)

N=1-

2

DT-1308939

-

30

44

40

39

51

-5

47

35

44

46

55

43

15

43

44

52

44

79

31

49

44

53

56

87

54

42

50

55

56

99

65

59

50

44

54

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

Cyta

rabi

ne (u

M)

0.1

2.3

71.

13.

39.

9

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

N=2

DT-1308940

-

-32

-44

-29

-16

13

35

6

4

-28

1

18

47

29

20

-8

8

10

52

36

7

-22

-4

13

56

46

20

-11

-10

12

63

53

25

3

-3

5


Cyta

rabi

ne (u

M)

0.1

2.3

71.

13.

39.

9

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th I

nhib

ition

(%

)

N=2-

3

DT-1308941

-

5

23

34

47

69

53

32

17

53

62

86

64

47

44

50

58

77

106

86

73

60

78

86

136

108

82

52

64

89

150

122

70

62

78

91


Topo

teca

n Hy

droc

hlor

ide

(u

M)

0.0

015

.004

6.0

14.0

41.1

2

Eribulin(E7389) (uM)0 .0012 .0037 .011 .033 .1

Grow

th In

hibi

tion

(%)

N=2

DT-1308942

-

7

26

74

92

96

89

89

91

93

97

96

108

110

111

101

97

102

115

117

121

112

105

97

133

125

126

103

102

104

159

152

145

113

107

118


Topo

teca

n Hy

droc

hlor

ide

(u

M)

0.0

14.0

41.1

2.3

71.

1

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th I

nhib

ition

(%

)

N=1-

2

DT-1308943

-

23

28

54

109

162

12

16

30

54

103

154

24

37

27

67

124

157

61

51

34

76

115

153

82

82

41

71

111

154

84

80

63

59

117

158

Dose MatrixNone | 100 | HT-1080 | ATP Lite | 72h |

Topo

teca

n Hy

droc

hlor

ide

(u

M)

0.1

2.3

71.

13.

39.

9

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

N=2

DT-1308944

-

59

88

115

144

190

22

71

88

117

152

186

57

74

91

118

158

191

96

78

92

125

166

190

151

84

95

129

156

194

180

90

108

145

168

193

Dose MatrixNone | 100 | KYSE-410 | ATP Lite | 72h |

Topo

teca

n Hy

droc

hlor

ide

(u

M)

0.0

14.0

41.1

2.3

71.

1

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th In

hibi

tion

(%)

N=2

DT-1308945

-

5

25

48

76

118

-5

-5

26

39

73

111

43

27

28

46

72

109

79

64

40

44

70

111

87

89

37

55

75

108

99

92

53

50

78

120


Topo

teca

n Hy

droc

hlor

ide

(u

M)

0.0

14.0

41.1

2.3

71.

1

Eribulin(E7389) (uM)0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037

Grow

th I

nhib

ition

(%

)

N=2-

3

DT-1308946

-

31

53

113

160

176

53

65

63

130

160

177

64

68

71

109

156

181

106

93

69

113

164

183

136

111

73

113

167

183

150

139

82

105

172

179


Topo

teca

n Hy

droc

hlor

ide

(u

M)

0.0

14.0

41.1

2.3

71.

1

Eribulin(E7389) (uM)0 1.4e-4 4.1e-4 .0012 .0037 .011

Grow

th In

hibi

tion

(%)

N=2

DT-1308947

-

53

73

92

123

146

20

56

67

90

119

143

63

51

66

102

122

141

121

90

82

93

136

149

130

100

91

106

127

148

130

100

98

118

136

154

MDA-MB-436 MDA-MB-468 SK-BR-3 KYSE-410 HT-1080 KATOIII

MDA-MB-468 NCI-H1650 NCI-H69 A2780 KYSE-410 HT-1080

Cytarabine

Topotecan


Eribulin + MCF

7

MD

A-M

B-43

6

MD

A-M

B-46

8

SK-B

R-3

A549

NCI

-H16

50

NCI

-H52

2

NCI

-H52

6

NCI

-H69

A278

0

SK-O

V-3

SNG

-M

KYSE

-410

FaD

u

MIA

PaC

a-2

PC-3

HT-

1080

KATO

III

MD

A-M

B-23

1

T47D

NCI

-H46

0

Hep

G2

786-

0

T24

A375

17-DMAG -3.35 0.10 -5.07 -5.95 -2.30 0.84 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

Cytarabine -3.92 -5.39 -8.73 -5.94 -1.32 -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

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.



016

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.

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.

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.

References1. Donoghue M, Lemery SJ, Yuan W, He K, Sridhara R, et al. (2012) Eribulin

Mesylate for the Treatment of Patients with Refractory Metastatic Breast Cancer: Use of a “Physician’s Choice” Control Arm in a Randomized Approval Trial. Clin Cancer Res 18: 1496-505.

2. Shetty N, Gupta S (2014) Eribulin drug review. South Asian J Cancer 3: 57-59.

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 49:1331–1337.

4. Towle MJ, Salvato KA, Budrow J, Wels BF, Kuznetsov G, et al. (2001) In vitro and in vivo anticancer activities of synthetic macrocyclic ketone analogs of Halichondrin B. Cancer Res 61: 1013-1021.

5. Schöffski P, Ray-Coquard IL, Cioffi A, Bui NB, Bauer S, et al. (2011) Activity of eribulin mesylate in patients with soft-tissue sarcoma: a phase 2 study in four independent histological subtypes. Lancet Oncol 12: 1045-1052.

6. Swami U, Chaudhary I, Ghalib MH, Goel S. (2012) Eribulin--A review of preclinical and clinical studies. Crit Rev Oncol Hemat 81:163–184.

7. Towle MJ, Nomoto K, Asano M, Kishi Y, Yu MJ, et al. (2012) Broad Spectrum Preclinical Antitumor Activity of Eribulin (Halaven®): Optimal Effectiveness under Intermittent Dosing Conditions. Anticancer Research 32: 1611-1620.

8. Funahashi Y, Okamoto K, Nomoto K, ODA Y, et al. (2014) Eribulin mesylate reduces tumor microenvironment abnormality by vascular remodeling in preclinical human breast cancer models. Cancer Sci 105:1334-1342.

9. Polastro L, Aftimos PG, Awada A (2014) Eribulin mesylate in the management of metastatic breast cancer and other solid cancers: a drug review. Expert Rev Anticancer Ther 14: 649-665.

10. Scarpace SL (2012) Eribulin mesylate (E7389): review of efficacy and tolerability in breast, pancreatic, head and neck, and non-small cell lung cancer. Clin Ther 34: 1467-1473.

11. Waller CF, Vynnychenko I, Bondarenko I, Shparyk Y, Hodge JP, et al. (2015) An Open-Label, Multicenter, Randomized Phase Ib/II Study of Eribulin Mesylate Administered in Combination With Pemetrexed Versus Pemetrexed Alone as Second-Line Therapy in Patients With Advanced Nonsquamous Non-Small-Cell Lung Cancer. Clin Lung Cancer 16: 92-99.

12. Schöffski P, Maki RG, Italiano A, Gelderblom H, Grignani G, et al. (2015) Randomized, open-label, multicenter, phase III study of eribulin versus dacarbazine in patients (pts) with leiomyosarcoma (LMS) and adipocytic sarcoma (ADI). J Clin Oncol 33: LBA10502.

13. Barrentina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, et al. (2012) The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483: 603-607.

14. Saiki AY, Caenepeel S, Yu D, Lofgren JA, Osgood T, et al. (2014) MDM2 antagonists synergize broadly and robustly with compounds targeting fundamental oncogenic signaling pathways. Oncotarget 5: 2030-2043.

15. Lehar J, Zimmermann GR, Krueger AS, Molnar RA, Ledell JY, et al. (2007) Chemical combination effects predict connectivity in biological systems. Mol Syst Biol 3:80

16. Lehar J, Krueger AS, Avery W, Heilbut AM, Johansen LM, et al. (2009) Synergistic drug combinations tend to improve therapeutically relevant selectivity. Nat Biotechnol 27: 659-666.

17. Jimeno A (2009) Eribulin: Rediscovering tubulin as an anticancer agent. Clin Cancer Res 15: 3903-3905.

18. Sharma SV, Bell DW, Settleman J, Haber DA (2007) Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 7: 169-181.

19. Lapatinib product insert:

20. Schaefer G, Shao L, Totpal K, Akita RW (2007) Erlotinib directly inhibits HER2 kinase activation and downstream signaling events in intact cells lacking epidermal growth factor receptor expression. Cancer Res 67: 1228-1238.

21. Li D, Ambrogio L, Shimamura T, Kubo S, Takahashi M, et al. (2008) BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models Oncogene 27: 4702–4711.

22. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, et al. (2011) Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 364: 2507-2516.

http://www.ncbi.nlm.nih.gov/pubmed/22282463


































http://hwmaint.meeting.ascopubs.org/cgi/content/abstract/33/18_suppl/LBA10502
























http://www.ncbi.nlm.nih.gov/pubmed/18408761http:/www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=18408761








017

23. Smalley KS, Sondak VK. (2010) Melanoma--an unlikely poster child for personalized cancer therapy. N Engl J Med 363: 876-878.

24. Nazarian R, Shi H, Wang Q, Kong X, Koya RC, et al. (2010) Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature 468: 973-977.

25. Johannessen CM, Boehm JS, Kim SY, Thomas SR, Wardwell L, et al. (2010)COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 468: 968-972.

26. Villanueva J, Vultur A, Lee JT, Somasundaram R, Fukunaga-Kalabis M, et al. ( 2010) Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell 18: 683-695.

27. Wagle N, Emery C, Berger MF, Davis MJ, Sawyer A, et al. (2011) Dissecting Therapeutic Resistance to RAF Inhibition in Melanoma by Tumor Genomic Profiling. J Clin Oncol 29: 3085-3096.

28. Faivre S, Raymond E, Woynarowski JM, Cvitkovic E (1999) Supraadditive effect of 2’,2’-difluorodeoxycytidine (gemcitabine) in combination with oxaliplatin in human cancer cell lines. Cancer Chemotherapy and Pharmacology 44: 117-123.

29. Distefano M, Ferlini C, De Vincenzo R, Gaggini C, Mancuso S, et al. (2000) Antagonistic effect of the combination gemcitabine/topotecan in ovarian cancer cells. Oncol Res 12: 355-359.

30. Akutsu M, Furukawa Y, Tsunoda S, Izumi T, Ohmine K, et al. (2002) Schedule-dependent synergism and antagonism between methotrexate and cytarabine against leukemia cell lines in vitro. Leukemia 16: 1808-1817.

31. Budman DR, Calabro A, Kreis W (2002) synergistic and antagonistic combinations of drugs in human prostate cancer cell lines in vitro. Anticancer Drugs 13: 1011-1016.

32. Li T, Ling YH, Goldman ID, Perez-Soler R (2007) Schedule-dependent cytotoxic synergism of pemetrexed and erlotinib in human non-small cell lung cancer cells. Clin Cancer Res 13: 3413-3422.

33. Barone C, Landriscina M, Quirino M, Basso M, Pozzo C, et al. (2007) Schedule-dependent activity of 5-fluorouracil and irinotecan combination in the treatment of human colorectal cancer: in vitro evidence and a phase I dose-escalating clinical trial. Br J Cancer 96: 21-28.

34. Ikeda H, Taira N, Nogami T, Shien K, Okada M, et al. (2011) Combination treatment with fulvestrant and various cytotoxic agents (doxorubicin, paclitaxel, docetaxel, vinorelbine, and 5-fluorouracil) has a synergistic effect in estrogen receptor-positive breast cancer. Cancer Science 102: 2038-2042.

35. Ehrhardt H, Schrembs D, Moritz C, Wachter F, Haldar S, et al. (2011) Optimized anti-tumor effects of anthracyclines plus Vinca alkaloids using a novel, mechanism-based application schedule. Blood 118: 6123-6131.

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.











































Documents

Identification of Combinatorial Drugs that Synergistically Kill both