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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:
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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.
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
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.
Rickles, et al. (2015)
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
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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.
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.
Rickles, et al. (2015)
012
Eribulin-Sensitive Cell Lines Eribulin-Insensitve Cell Lines
Target Eribulin + MCF
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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
Dose MatrixNone | 100 | A2780 | 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-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
Loewe ExcessNone | 100 | A2780 | 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=
2.33
(.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
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.
Rickles, et al. (2015)
013
Eribulin-Sensitive Cell Lines Eribulin-Insensitve Cell Lines
Target 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
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
Dose Matrix | None | 100 | FaDu | ATP Lite | 72h
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
Dose Matrix | None | 100 | MDA-MB-468 | ATP Lite | 72h
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
Dose Matrix | None | 100 | T47D | ATP Lite | 72h
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
Dose Matrix | None | 500 | NCI-H69 | ATP Lite | 72h
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
Dose Matrix | None | 100 | A2780 | ATP Lite | 72h
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
Dose Matrix | None | 100 | HT-1080 | ATP Lite | 72h
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
Dose Matrix | None | 100 | A2780 | ATP Lite | 72h
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
Dose Matrix | None | 100 | HT-1080 | ATP Lite | 72h
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
Dose Matrix | None | 100 | FaDu | ATP Lite | 72h
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
Dose Matrix | None | 100 | MDA-MB-468 | ATP Lite | 72h
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
Dose Matrix | None | 500 | NCI-H69 | ATP Lite | 72h
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
Dose Matrix | None | 100 | T47D | ATP Lite | 72h
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
Dose MatrixNone | 500 | NCI-H69 | ATP Lite | 72h |
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
Dose MatrixNone | 100 | MDA-MB-468 | ATP Lite | 72h |
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
Dose MatrixNone | 100 | A2780 | ATP Lite | 72h |
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
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.
Rickles, et al. (2015)
014
Dose Matrix | 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
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
Dose Matrix | 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
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
Dose MatrixNone | 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
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
Dose MatrixNone | 100 | A2780 | ATP Lite | 72h |
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
Loewe ExcessNone | 100 | A2780 | ATP Lite | 72h |
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
Dose Matrix | 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
(%)
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
Dose Matrix | 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
(%)
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
Eribulin-Sensitive Cell Lines Eribulin-Insensitve Cell Lines
Target 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
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,
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.
Rickles, et al. (2015)
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
Dose Matrix | None | 100 | MDA-MB-436 | ATP Lite | 72h
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
Dose Matrix | None | 100 | HT-1080 | 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-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
Dose Matrix | None | 100 | MDA-MB-468 | 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=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 |
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
Dose MatrixNone | 100 | NCI-H1650 | 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 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
Dose MatrixNone | 100 | MDA-MB-468 | 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 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
Dose MatrixNone | 500 | NCI-H69 | 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 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-Sensitive Cell Lines Eribulin-Insensitve Cell Lines
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.
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.
Rickles, et al. (2015)
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.
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Copyright: © 2015 Rickles RJ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.