identification of combinatorial drugs that synergistically kill both eribulin-sensitive and...

8/19/2019 Identification of Combinatorial Drugs that Synergistically Kill both Eribulin-Sensitive and Eribulin- Insensitive Tumor …

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Global Journal of Cancer Therapy

Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identication of Combinatorial Drugs that Synergistically Kill both Eribulin-

Sensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.009

eertechz

Abst ract

Eribulin sensitivity was examined in a panel of twenty-ve 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 identied. In summary, the preclinical studies described here have

identied 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.

o care. Chemotherapeutic drugs are ofen most effective when given

in combination, in particular when synergistic killing is achieved

without additive toxicity. o date, potential combination therapies

or eribulin have been explored with only a limited number o anti-

cancer agents. We thereore have perormed an in vitro study o

eribulin combined with 34 anticancer agents in 25 different tumor cell

lines o various types, through use o a combination high throughput

screening (cHS) platorm. Our goals were to identiy drugs that

might be paired with eribulin to increase clinical efficacy in metastatic

breast cancer, to identiy drugs that convert eribulin non-responders

to responders, and to identiy new therapeutic indications or eribulin

utilization. Several compelling synergy effects with approved and

emerging drugs were observed. Te preclinical data provides insights

about uture clinical development strategies.

Materials and Methods

Materials

Cell lines were purchased rom European Collection o Cell

Cultures o Public Health England (A2780), Japanese Collection o

Research Bioresouces Cell Bank o the National Institute o Biomedical

Innovation (SNG-M), German Collection o Microorganisms and

Cell Cultures (KYSE-410), and American ype Culture Collection

(all other cell lines). All cell lines are included in the Cancer Cell Line

Encyclopedia [13]. Cell culture media, etal bovine serum, and cell

culture supplements were purchased rom Gibco Lie echnologies

(Termo Scientific). Chemical matter was purchased rom Enzo Lie

Sciences, Inc., Micro Source Discovery Systems, Sigma-Aldrich Co.

LLC, Selleck Chemicals, Sequoia Research Products Limited, and

Introduction

Eribulin mesylate, a microtubule dynamics inhibitor with a

mechanism distinct rom most other anti-tubulin therapeutics, isapproved in the United States and many other countries or treatment

o certain patients with advanced or metastatic breast cancer [1,2].

Eribulin works by binding to exposed beta-tubulin subunits at the

plus ends o microtubules, where it acts through end-poisoning

mechanisms to inhibit microtubule growth and sequester tubulin

into non-unctional 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 o

preclinical cancer models; as a result, numerous clinical trials o its

effectiveness under monotherapy conditions are ongoing in other

non-breast tumor types [4-9]. Although eribulin ailed to showmeaningul activities in clinical trials o 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

sof tissue sarcoma compared with dacarbazine [12], pointing to

additional clinical uses or eribulin. Clinical trials are ongoing to

evaluate eribulin effectiveness or treatment o 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 o other chemotherapies: monotherapy benefits tend

to be limited and it is ofen difficult to surpass the benefits o standard

Research Article

Identification of CombinatorialDrugs 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 Ofcer), E-mail:

www.peertechz.com


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Sensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.

Rickles, et al. (2015)

010

oronto Research Chemicals. Cell prolieration was measured by

AP 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 ChaliceM Analyzer sofware.

Combination High-Throughput Screening

Cells were seeded in 384-well and 1536-well tissue culture treated

assay plates at cell densities ranging rom 100 to 500 cells per well.

wenty-our hours afer cell seeding, compounds were added to

assay plates with multiple replicates. Compounds were added to cells

using a 6x6 dose matrix, ormed by six treatment points (including

DMSO control) o Eribulin and each combination partner as single

agents and twenty-five combination points. Please see reerence 12

or additional detail. Concentration sampling ranges were selected

afer review o single agent activity o each molecule across the cell

line panel. Generally, concentrations between the EC10 and EC90

were sampled.

At the time o treatment, a set o assay plates (which do not

receive treatment) were collected and AP levels were measured

by adding APLite. Tese Vehicle-zero (V0) plates were measured

using an EnVision Multilabel Reader (Perkin Elmer). reated assay

plates were incubated with compound or seventy-two hours. Afer

seventy-two hours, treated assay plates were developed or endpoint

analysis using APLite. All data points were collected via automatedprocesses; quality controlled; and analyzed using Horizon’s

proprietary sofware.

Horizon utilizes Growth Inhibition (GI) as a measure o cell

viability. Te cell viability o vehicle is measured at the time o dosing

(V0) and afer seventy-two hours (V). A GI reading o 0% represents

no growth inhibition - cells treated with compound () 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 or compounds reaching

a plateau at this effect level. A GI 200% represents complete death o

all cells in the culture well. Compounds reaching an activity plateau o

GI 200% are considered cytotoxic. Horizon calculates GI by applying

the ollowing test and equation:

00

0

00

0

If : :

If

100 *(1 )

100*( : : 1 )

T V V T

V

T T

V V

V V

−< −

−≥ −

−

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

control measure afer seventy-two hours, and Vo is the vehicle control

measure at time zero.

Analysi s of combinat ion screening resul ts

o measure combination effects in excess o Loewe additivity,

Horizon has devised a scalar measure to characterize the strength o

synergistic interaction termed the Synergy Score [13,14]. Te Synergy

Score equation integrates the experimentally-observed activity

volume at each point in the matrix in excess o a model surace

numerically derived rom the activity o the component agents using

the Loewe model or additivity. Additional terms in the SynergyScore equation are used to normalize or various dilution actors used

or individual agents and to allow or comparison o synergy scores

across an entire experiment.

Loewe Volume is an additional combination model score used to

assess the overall magnitude o the combination interaction in excess

o the Loewe additivity model. Loewe Volume is particularly useul

when distinguishing synergistic increases in a phenotypic activity

(positive Loewe Volume) versus synergistic antagonisms (negative

Loewe Volume). Please see reerence 13 or more detail.

Self-cross based combination screening analysis

In order to objectively establish hit criteria or the combinationscreen analysis, twelve compounds were selected to be sel-crossed

across the twenty-five cell line panel as a means to empirically

determine a baseline additive, non-synergistic response. Te identity

o the twelve sel-cross compounds was determined by selecting

compounds with a variety o maximum response values and single

agent dose response steepness. Tose drug combinations which

yielded effect levels that statistically superseded those baseline

additivity values were considered synergistic. Te Synergy Score

measure was used or the sel-cross analysis. Synergy Scores o sel-

crosses are expected to be additive by definition and, thereore,

maintain a synergy score o zero. However, while some sel-cross

Synergy Scores are near zero, many are greater suggesting that

experimental noise or non-optimal curve fitting o the single agentdose 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 or the sel-cross based combination screen analysis, ocusing

on sel-cross behavior in individual cell lines versus global review o

the cell line panel activity. Combinations where the Synergy Score is

greater than the mean sel-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.

Results

Evaluation of eribulin potency using a cell-based

assayEribulin’s antiprolierative activity was assessed in a panel o

twenty-five human cancer cells lines representing a variety o tumor

types using a three-old, ten-point dose titration o drug. Cells were

incubated with drug or 72 hours. Cellular AP levels were quantified

as a measurement o effects on prolieration. A measurement was

perormed at the time o drug addition (time zero, 0) in order to

calculate a Growth Inhibition percentage, providing inormation

about cytostatic and cytotoxic activities. Cell lines were designated as

sensitive to eribulin i the GI50 values were


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011

in Figure 1 were included in the subsequent combination screen,

with the ollowing rationale: inclusion o eribulin sensitive cell lines

would provide models to identiy drugs with the potential to increase

eribulin efficacy, while inclusion o insensitive lines would acilitateidentification o drugs capable o converting eribulin insensitive cells

to eribulin sensitive cells.

Receptor tyrosine kinase target-based clusteranalysis

Te thirty-five compound enhancer library (Supplemental able

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 or the combination screen analysis, twelve compounds were

selected to be sel-crossed across the twenty-five cell line panel as a

means to empirically determine a baseline additive, non-synergistic

response. Te identity o the twelve sel-cross compounds was

determined by selecting compounds with a variety o maximum

response values and single agent dose response steepness. Tose drug

combinations which yielded effect levels that statistically superseded

those baseline additivity values were considered synergistic.

A summary matrix view heat map o combination drug Synergy

Scores which exceed the cell line specific sel-cross thresholds at

2σ’s above the mean is shown in Supplemental Figure 1. Using this

strategy, 19.7% o the combinations exhibited some synergistic

activity at the 95% confidence level. Using a more stringent criteria

o 3σ cutoff (99% confidence), synergy was observed or 12.5% o the

combinations (Supplemental Figure 2).

Te 35 compound enhancer library utilized contains some

redundancy in terms o targets and pathway inhibitors. Whenever

possible, target inormation was used to cluster similarly annotated

enhancers across the cell line panel, and the Loewe Excess values

or Growth Inhibition matrices with high-to-moderate synergy

scores were individually reviewed to evaluate combination activities.

Te strategy o using target or pathway cluster profiles with visual

inspection o matrices provides additional evidence linking a

particular target with combination activity, and helps to highlight

subtle synergies that might otherwise be overlooked or insufficientlyrevealed due to steep single agent dose response curves. A Synergy

Score heat map or select target/pathway clusters is displayed in

Figure 2, with cell lines clustered first according to eribulin sensitivity/

insensitivity then urther demarcated based on tumor type.

As an example o 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

o combination activity. Te mechanism o action o BIBW-2992

(irreversible inhibition) and higher potency or ErbB1 and ErbB2

may explain the greater breath-o-activity. Alternatively, synergies

observed with BIBW-2992 but not lapatinib or erlotinib may be

the result o unique polypharmacy with BIBW-2992 inhibiting

secondary targets not affected by the other ErbB1/ErbB2 inhibitors.

Representative Growth Inhibition and Loewe Excess dose matrices

or some o the lapatinib combinations are shown in Figure 3 with

examples o erlotinib and BIBW-2992 combination activities shown

in Supplemental Figures 3, 4. Low micromolar concentrations o

lapatinib and erlotinib have been observed in pharmacokinetic

studies [18,19] suggesting the potential or reproducing the observed

preclinical synergy here in a clinical setting.

PI3K pathway inhibi tors

Dysregulation o the PI3K pathway can transorm cells by virtue

o constitutive activation and ultimately, stimulation o cellular

prolieration and suppression o pro-apoptotic signaling. Four PI3K

pathway inhibitors were included in the screen. AZD8055 is a potent,

selective, and orally bioavailable AP-competitive mOR kinase

inhibitor (ORC1 and ORC2); everolimus, an allosteric mOR

(ORC1) inhibitor; BEZ235, a dual pan-PI3K/mOR inhibitor; and

M C F 7

M D A - M B - 2 3 1

M D A - M B - 4 3 6

M D A - M B - 4 6 8

S K - B R - 3

T 4 7 D

A 5 4 9

N C I - H 1 6 5 0

N C I - H 4 6 0

N C I - H 5 2 2

N C I - H 5 2 6

N C I - H 6 9

A 2 7 8 0

S K - O V - 3

S N G - M

H e p

G 2

K Y S E - 4 1 0

F a D u

7 8 6 - 0

T 2 4

M I A P a C a - 2

P C - 3

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H T - 1 0 8 0

K A T O I

I I0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

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5.0015.0025.00

35.00

E r i b u l i n G I 5 0

( n M

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B r e a s t

L u n g

O v a r i a n

E n d o m e t r i a l

L i v e r

E s o p h a g e a l

H e a d + N e c k

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M e l a n o m a

F i b r o s a r c o m a

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H e p

G 2

T 2 4

M D A - M B - 2 3 1

N C I - H 4 6 0

7 8 6 - 0

T 4 7 D

A 3 7 5

A 2 7 8 0

K A T O I I I

F a D u

N C I - H 1 6 5 0

A 5 4 9

M C F 7

N C I - H 5 2 6

S N G - M

N C I - H 5 2 2

N C I - H 6 9

M D A - M B - 4 3 6

K Y S E - 4 1 0

P C - 3

M I A P a C a - 2

H T - 1 0 8 0

S K - B R - 3

S K - O V - 3

M D A - M B - 4 6 8

0.0

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0.6

0.7

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( n M

)

Figure 1: Assessment of eribulin activity in twenty-ve 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-ve cell line panel is 0.51 nM.

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

response and extended PFS to 5.3 months in V600E melanomapatients in the BRIM3 study [21], in a first-line BRAF mutated

melanoma therapy. However, the treatment o vemuraenib caused

resistance in some patients through its 2-18 months post treatment

[22]. Recently, our articles reported five resistance mechanisms

or vemuraenib [23-27]. Tose authors attributed their resistance

mechanisms to the overexpression o PDGFR-β tyrosine kinase [23],

and/or Q61K NRAS [24], CO 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 useul to

find out appropriate combination partners.

In this study, we describe the identification o approved and

emerging drugs that synergize when combined with eribulin, with

good breadth o combination activity observed in the twenty five cell

line panel used or screening. Synergy is observed in both eribulin-

sensitive and insensitive cell lines, suggesting potential clinical benefit

or both responders and non-responders o eribulin monotherapy.

In addition to approved drugs that target EGFR/HER2 (erlotinib,

lapatinib, BIBW2992/aatinib), mOR (everolimus), and MEK

(trametinib), we show that emerging drugs such as BEZ235 (pan-

PI3K/mOR), BKM-120 (PI3Kα), and AB-263 (BCL-2 amily

inhibitor) strongly synergize with eribulin in certain cell lines.

Furthermore, the use o both target-specific (EGFR/HER2, MEK)

and pathway-specific (PI3K), mechanistically redundant compounds

in our studies validates the identified target specificities as being

important or synergy. By evaluating a wide concentration rangeor 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 ound 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 o in vitro drug activities is that

patterns o 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. o

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 xenograf models in

mice, with these combinations showing acceptable toxicity profiles

(manuscript in preparation).

An important goal or any monotherapy or drug combination is

the identification o predictors o response so that clinicians can select

the patient population most likely to benefit rom treatment. Te

screen described here represents the discovery phase o such a project

and additional work is required to identiy predictors o response with

a certainty that can drive patient selection. For instance, the kinetics

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

C y t a r a b i n e ( u M )

0

. 0 1 4 . 0

4 1

. 1 2

. 3 7

1 . 1

Eribulin(E7389) (uM)

0 1.4e-44.1e-4 .0012 .0037 .011

G r o w t h I n h i b i t i o n ( % )

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

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


0

. 0 1 4 . 0

4 1

. 1 2

. 3 7

1 . 1


0 4.6e-51.4e-4 4.1e-4 .0012 .0037


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


0

. 0 1 4 . 0

4 1

. 1 2

. 3 7

1 . 1


0 4.6e-51.4e-4 4.1e-4 .0012 .0037


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 | KY SE-410 | ATP Lite | 72h


0

. 1 2

. 3 7

1 . 1

3 . 3

9 . 9


0 4.6e-51.4e-4 4.1e-4 .0012 .0037


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


0

. 1 2

. 3 7

1 . 1

3 . 3

9 . 9


0 1.4e-44.1e-4 .0012 .0037 .011


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

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


0

. 1 2

. 3 7

1 . 1

3 . 3

9 . 9


0 4.6e-51.4e-4 4.1e-4 .0012 .0037


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

Dose MatrixNone | 100 | A2780

| ATP Lite | 72h |

T o p o t e c a n H y d r o c h l o r i d e

( u M )

0

. 0 0 1 5 . 0

0 4 6

. 0 1 4 . 0

4 1

. 1 2

Eribulin(E7389) (uM)0 .0012.0037 . 01 1 . 0 33 . 1


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

Dose MatrixNone | 100 | NCI-

H1650 | ATP Lite | 72h |


( u M )

0

. 0 1 4 . 0

4 1

. 1 2

. 3 7

1 . 1

Eribulin(E7389) (uM)0 1.4e-44.1e-4 .0012 .0037 .011


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 |


( u M )

0

. 1 2

. 3 7

1 . 1

3 . 3

9 . 9

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


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 |


( u M )

0

. 0 1 4 . 0

4 1

. 1 2

. 3 7

1 . 1

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


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

Dose MatrixNone | 100 | MDA-

MB-468 | ATP Lite | 72h |


( u M )

0

. 0 1 4 . 0

4 1

. 1 2

. 3 7

1 . 1

Eribulin(E7389) (uM)0 4.6e-51.4e-4 4.1e-4 .0012 .0037


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

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


( u M )

0

. 0 1 4 . 0

4 1

. 1 2

. 3 7

1 . 1

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


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-Sensitve Cell Lines Eribulin-Insensitve Cell Lines

Eribulin + M C F

7

M D A

- M B - 4 3 6

M D A

- M B - 4 6 8

S K - B

R - 3

A 5 4 9

N C I - H 1 6 5 0

N C I - H 5 2 2

N C I - H 5 2 6

N C I - H 6 9

A 2 7 8

0

S K - O

V - 3

S N G - M

K Y S E

- 4 1 0

F a D u

M I A

P a C a - 2

P C - 3

H T - 1

0 8 0

K A T O I I I

M D A

- M B - 2 3 1

T 4 7 D

N C I - H 4 6 0

H e p

G 2

7 8 6 - 0

T 2 4

A 3 7 5

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 -1.5 2 -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 - 0.1 7 1 .1 4 0 .3 1 - 0.8 8 - 0.6 8

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 compoun ds screened in combination wi th 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.


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016

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

relationships, i any, o synergy/non-synergy to intrinsic or acquired

resistance should be urther defined. o power such analyses,

additional cell lines could be screened to generate a larger data set

or more detailed genetic characterization. Additional analyses using

larger data sets with sufficient breadth o known genetic profiles will

be required to generate a ully vetted understanding o response

prediction.

In addition to identiying drug synergies, we have also identified

antagonistic combinations. For example, low concentrations o

cytarabine or topotecan have little effect on prolieration as single

agents, but can dramatically antagonize eribulin activity under

combination conditions. Indeed, this is not without precedent:

antagonism has been observed or a variety o cancer drug

combinations in preclinical studies, prompting ollow up analyses

to investigate both the mechanisms o the synergies as well as the

effects o drug combination sequencing [28-35]. For instance, in

one extensively studied example, the anthracycline doxorubicin

antagonized activity o the vinca alkaloid vincristine in 83% (15/18)

o hematopoietic cell lines tested. Tis antagonism was reproduced

in vivo in a xenograf model and was also observed in 34% (12/35) o

childhood leukemia cells simultaneously treated with both drugs ex

vivo [35]. Moreover, combination activity was shown to be sequence

dependent: i cells are exposed to the anthracycline first, antagonism

is observed, but i exposure to vinca alkaloid precedes anthracycline,

the combination result is beneficial. A p53-dependent mechanism or

this antagonism was proposed, where anthracycline effects stabilize

anti-apoptotic BCL2 amily members, thus reducing cytotoxic effectso 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 unctional as was

the case in the preceding example, but urther 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 or selective, synergistic tumor cell killing.

In order to utilize the current results with the goal o clinical

implementation, urther studies, including sequencing o drugsand both in vivo efficacy and toxicology studies will be needed. Our

results suggest that urther 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 o

a combination drug regimen in patients in the context o controlled

clinical trials.

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