Global Journal of Cancer Therapy Richard J Rickles1, Junji Matsui3, Ping Zhu1, Yasuhiro Funahashi2,3, Jill M Grenier1, Janine Steiger1, Nanding Zhao2, Bruce A Littlefield2, Kenichi Nomoto2,3 and Toshimitsu Uenaka2* Horizon Discovery Inc., MA, Japan Eisai Inc., MA, Japan 3 Eisai Co., Ltd., Japan 1 2
Dates: Received: 17 October, 2015; Accepted: 03 November, 2015; Published: 04 November, 2015 *Corresponding author: Toshimitsu Uenaka, Ph.D., Executive Director, Production Creation Headquarter, Oncology & Antibody Drug Strategy, CINO (Chief Innovation Officer), E-mail: www.peertechz.com
eertechz
Research Article
Identification of Combinatorial Drugs that Synergistically Kill both Eribulin-Sensitive and EribulinInsensitive Tumor Cells Abstract Eribulin sensitivity was examined in a panel of twenty-five human cancer cell lines representing a variety of tumor types, with a preponderance of breast and lung cancer cell lines. As expected, the cell lines vary in sensitivity to eribulin at clinically relevant concentrations. To identify combination drugs capable of increasing anticancer effects in patients already responsive to eribulin, as well as inducing de novo anticancer effects in non-responders, we performed a combinatorial high throughput screen to identify drugs that combine with eribulin to selectively kill tumor cells. Among other observations, we found that inhibitors of ErbB1/ErbB2 (lapatinib, BIBW-2992, erlotinib), MEK (E6201, trametinib), PI3K (BKM-120), mTOR (AZD 8055, everolimus), PI3K/mTOR (BEZ 235), and a BCL2 family antagonist (ABT-263) show combinatorial activity with eribulin. In addition, antagonistic pairings with other agents, such as a topoisomerase I inhibitor (topotecan hydrochloride), an HSP-90 inhibitor (17-DMAG), and gemcitabine and cytarabine, were identified. In summary, the preclinical studies described here have identified several combination drugs that have the potential to either augment or antagonize eribulin’s anticancer activity. Further elucidation of the mechanisms responsible for such interactions may be important for identifying valuable therapeutic partners for eribulin.
Introduction Eribulin mesylate, a microtubule dynamics inhibitor with a mechanism distinct from most other anti-tubulin therapeutics, is approved in the United States and many other countries for treatment of certain patients with advanced or metastatic breast cancer [1,2]. Eribulin works by binding to exposed beta-tubulin subunits at the plus ends of microtubules, where it acts through end-poisoning mechanisms to inhibit microtubule growth and sequester tubulin into non-functional aggregates. In mitosis, this results in G2/M cell cycle arrest, irreversible mitotic blockade, and ultimately cell death by apoptosis [3]. Eribulin has broad anticancer activity in a wide variety of preclinical cancer models; as a result, numerous clinical trials of its effectiveness under monotherapy conditions are ongoing in other non-breast tumor types [4-9]. Although eribulin failed to show meaningful activities in clinical trials of head and neck, pancreatic and advanced non-small cell lung cancer [10,11], improvements in overall survival by eribulin were reported in a 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
of care. Chemotherapeutic drugs are often most effective when given in combination, in particular when synergistic killing is achieved without additive toxicity. To date, potential combination therapies for eribulin have been explored with only a limited number of anticancer agents. We therefore have performed an in vitro study of eribulin combined with 34 anticancer agents in 25 different tumor cell lines of various types, through use of a combination high throughput screening (cHTS) platform. Our goals were to identify drugs that might be paired with eribulin to increase clinical efficacy in metastatic breast cancer, to identify drugs that convert eribulin non-responders to responders, and to identify new therapeutic indications for eribulin utilization. Several compelling synergy effects with approved and emerging drugs were observed. The preclinical data provides insights about future clinical development strategies.
Materials and Methods Materials Cell lines were purchased from European Collection of Cell Cultures of Public Health England (A2780), Japanese Collection of Research Bioresouces Cell Bank of the National Institute of Biomedical Innovation (SNG-M), German Collection of Microorganisms and Cell Cultures (KYSE-410), and American Type Culture Collection (all other cell lines). All cell lines are included in the Cancer Cell Line Encyclopedia [13]. Cell culture media, fetal bovine serum, and cell culture supplements were purchased from Gibco Life Technologies (Thermo Scientific). Chemical matter was purchased from Enzo Life Sciences, Inc., Micro Source Discovery Systems, Sigma-Aldrich Co. LLC, Selleck Chemicals, Sequoia Research Products Limited, and
Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identification of Combinatorial Drugs that Synergistically Kill both EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.
009
Rickles, et al. (2015)
Toronto Research Chemicals. Cell proliferation was measured by ATP Lite 1step Luminescence Assay System (Perkin Elmer).
Methods Cells were thawed and grown in culture media according to 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 Screening Cells were seeded in 384-well and 1536-well tissue culture treated assay plates at cell densities ranging from 100 to 500 cells per well. Twenty-four hours after cell seeding, compounds were added to assay plates with multiple replicates. Compounds were added to cells using a 6x6 dose matrix, formed by six treatment points (including DMSO control) of Eribulin and each combination partner as single agents and twenty-five combination points. Please see reference 12 for additional detail. Concentration sampling ranges were selected after review of single agent activity of each molecule across the cell line panel. Generally, concentrations between the EC10 and EC90 were sampled. At the time of treatment, a set of assay plates (which do not receive treatment) were collected and ATP levels were measured by adding ATPLite. These Vehicle-zero (V0) plates were measured using an EnVision Multilabel Reader (Perkin Elmer). Treated assay plates were incubated with compound for seventy-two hours. After seventy-two hours, treated assay plates were developed for endpoint analysis using ATPLite. All data points were collected via automated processes; quality controlled; and analyzed using 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: If T :< V0 : 100 * (1 −
T − V0 ) V0
If T :≥ V0 : 100 * (1 −
T − V0 ) V − V0
Where T is the signal measure for a test article, V is the vehicle-treated control measure after seventy-two hours, and Vo is the vehicle control measure at time zero.
Analysis of combination screening results To measure combination effects in excess of Loewe additivity, Horizon has devised a scalar measure to characterize the strength of synergistic interaction termed the Synergy Score [13,14]. The Synergy
010
Score equation integrates the experimentally-observed activity volume at each point in the matrix in excess of a model surface numerically derived from the activity of the component agents using the Loewe model for additivity. Additional terms in the Synergy Score equation are used to normalize for various dilution factors used for individual agents and to allow for comparison of synergy scores across an entire experiment. Loewe Volume is an additional combination model score used to assess the overall magnitude of the combination interaction in excess of the Loewe additivity model. Loewe Volume is particularly useful when distinguishing synergistic increases in a phenotypic activity (positive Loewe Volume) versus synergistic antagonisms (negative Loewe Volume). Please see reference 13 for more detail.
Self-cross based combination screening analysis In order to objectively establish hit criteria for the combination screen analysis, twelve compounds were selected to be self-crossed across the twenty-five cell line panel as a means to empirically determine a baseline additive, non-synergistic response. The identity of the twelve self-cross compounds was determined by selecting compounds with a variety of maximum response values and single agent dose response steepness. Those drug combinations which yielded effect levels that statistically superseded those baseline additivity values were considered synergistic. The Synergy Score measure was used for the self-cross analysis. Synergy Scores of selfcrosses are expected to be additive by definition and, therefore, maintain a synergy score of zero. However, while some self-cross Synergy Scores are near zero, many are greater suggesting that experimental noise or non-optimal curve fitting of the single agent dose responses are contributing to the slight perturbations in the score. Given the potential differences in cell line sensitivity to the eribulin combination activities, we chose to use a cell-line centric strategy for the self-cross based combination screen analysis, focusing on self-cross behavior in individual cell lines versus global review of the cell line panel activity. Combinations where the Synergy Score is greater than the mean self-cross plus two standard deviations (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 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 EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.
Rickles, et al. (2015)
in Figure 1 were included in the subsequent combination screen, with the following rationale: inclusion of eribulin sensitive cell lines would provide models to identify drugs with the potential to increase eribulin efficacy, while inclusion of insensitive lines would facilitate identification of drugs capable of converting eribulin insensitive cells to eribulin sensitive cells.
Receptor tyrosine kinase target-based cluster analysis The thirty-five compound enhancer library (Supplemental Table 1) was screened in combination with eribulin in the twenty-five cell lines described above in Figure 1. In order to objectively establish hit criteria for the combination screen analysis, twelve compounds were selected to be self-crossed across the twenty-five cell line panel as a means to empirically determine a baseline additive, non-synergistic response. The identity of the twelve self-cross compounds was determined by selecting compounds with a variety of maximum response values and single agent dose response steepness. Those drug combinations which yielded effect levels that statistically superseded those baseline additivity values were considered synergistic. A summary matrix view heat map of combination drug Synergy Scores which exceed the cell line specific self-cross thresholds at 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.
As an example of enhancer library mechanistic redundancy, the combination screen contained three drugs known to inhibit ErbB1/ErbB2 (EGFR/HER2) in cell-based assays: lapatinib, BIBW2992 and erlotinib [18-21]. BIBW-2992 exhibited the best breadth of combination activity. The mechanism of action of BIBW-2992 (irreversible inhibition) and higher potency for ErbB1 and ErbB2 may explain the greater breath-of-activity. Alternatively, synergies observed with BIBW-2992 but not lapatinib or erlotinib may be the result of unique polypharmacy with BIBW-2992 inhibiting secondary targets not affected by the other ErbB1/ErbB2 inhibitors. Representative Growth Inhibition and Loewe Excess dose matrices for some of the lapatinib combinations are shown in Figure 3 with examples of erlotinib and BIBW-2992 combination activities shown in Supplemental Figures 3, 4. Low micromolar concentrations of lapatinib and erlotinib have been observed in pharmacokinetic studies [18,19] suggesting the potential for reproducing the observed preclinical synergy here in a clinical setting.
PI3K pathway inhibitors Dysregulation of the PI3K pathway can transform cells by virtue of constitutive activation and ultimately, stimulation of cellular proliferation and suppression of pro-apoptotic signaling. Four PI3K pathway inhibitors were included in the screen. AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mTOR kinase inhibitor (TORC1 and TORC2); everolimus, an allosteric mTOR (TORC1) inhibitor; BEZ235, a dual pan-PI3K/mTOR inhibitor; and 35.00 25.00 15.00 5.00 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
Ovarian
Endometrial Liver Esophageal Head+Neck Kidney Bladder Pancreas Prostate Melanoma Fibrosarcoma Stomach
Lung
Breast
Hep G2 T24 MDA-MB-231 NCI-H460 786-0 T47D A375 A2780 KATOIII FaDu NCI-H1650 A549 MCF7 NCI-H526 SNG-M NCI-H522 NCI-H69 MDA-MB-436 KYSE-410 PC-3 MIA PaCa-2 HT-1080 SK-BR-3 SK-OV-3 MDA-MB-468
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
MCF7 MDA-MB-231 MDA-MB-436 MDA-MB-468 SK-BR-3 T47D A549 NCI-H1650 NCI-H460 NCI-H522 NCI-H526 NCI-H69 A2780 SK-OV-3 SNG-M Hep G2 KYSE-410 FaDu 786-0 T24 MIA PaCa-2 PC-3 A375 HT-1080 KATO III
Eribulin GI5 0 (nM )
35.00 25.00 15.00 5.00
Eribulin GI 5 0 (nM )
The strategy of using target or pathway cluster profiles with visual inspection of matrices provides additional evidence linking a particular target with combination activity, and helps to highlight subtle synergies that might otherwise be overlooked or insufficiently revealed due to steep single agent dose response curves. A Synergy Score heat map for select target/pathway clusters is displayed in Figure 2, with cell lines clustered first according to eribulin sensitivity/ insensitivity then further demarcated based on tumor type.
Figure 1: Assessment of eribulin activity in twenty-five cell lines. Bar graph display of GI50 values based on relative sensitivity (Waterfall plot, left) or after clustering cell lines based on tumor type (right). Cells were exposed to eribulin for 72 hours. The assay endpoint was measurement of ATP (as a surrogate for effects on proliferation). The median GI50 for the twenty-five cell line panel is 0.51 nM.
011
Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identification of Combinatorial Drugs that Synergistically Kill both EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.
Rickles, et al. (2015)
PI3K Pathway
MEK
NCI-H522
NCI-H69
A2780
SNG-M
KYSE-410
MDA-MB-231
T47D
NCI-H460
Hep G2
786-0
T24
A375
9.00
6.21
6.78
6.74
6.28
3.02
5.01
18.61
7.59
9.83
16.05
7.77
6.22
5.05
4.20
1.45
4.90
12.25
17.59
8.52
11.31
8.63
12.25
3.24
12.15
1.26
2.96
3.25
4.90
2.49
3.91
5.50
3.94
4.38
7.25
23.98
1.49
2.10
1.18
4.36
0.96
2.99
7.77
5.48
7.34
6.49
6.74 11.77
KATOIII
PC-3
FaDu
HT-1080
NCI-H1650
1.80
3.14
MIA PaCa-2
A549
1.31
Erlotinib
SK-OV-3
SK-BR-3
BIBW-2992
NCI-H526
MDA-MB-468
ErbB1/ErbB2
Eribulin +
MDA-MB-436
Target
Eribulin-Insensitve Cell Lines
MCF7
Eribulin-Sensitive Cell Lines
Lapatinib
7.86
1.77
9.33
2.35
1.65
2.20
0.43
1.89
4.55
6.87
4.80
8.69
9.15
26.61
0.63
0.47
1.36
3.25
0.76
3.11
9.85
7.68
17.46
14.14
AZD 8055
11.84
5.04
8.60
1.13
7.71
6.53
1.39
2.96
8.19
17.12
7.62
5.44
9.68
9.38
1.53
8.79
10.47
6.44
4.14
7.65
10.15
1.70
2.85
4.48
5.37
BEZ235
8.03
5.32
16.92
1.32
6.78
5.92
2.86
2.34
7.90
10.80
4.43
12.44
9.16
18.70
2.36
8.18
9.60
4.34
7.28
13.32 12.30
10.10
4.16
6.75
8.36
BKM-120
6.12
2.87
6.05
1.13
5.38
2.82
1.20
3.71
5.47
10.76
3.27
7.03
4.86
7.72
0.75
3.02
8.11
2.79
2.10
7.17
5.99
3.22
2.49
2.34
1.45
Everolimus
4.69
2.78
11.76
2.56
1.56
5.84
1.50
4.05
7.14
6.97
4.84
4.14
5.08
5.71
1.02
4.24
9.81
3.92
2.88
7.07
1.97
1.85
1.60
1.56
4.42
Trametinib
1.32
3.98
10.18
0.50
5.63
3.66
0.26
0.87
2.09
14.55
4.80
0.71
4.10
4.76
3.87
0.88
0.72
1.69
14.92
1.58
9.31
10.41
3.65
2.90
1.10
E6201
0.71
1.72
2.19
0.00
7.06
4.05
1.02
1.61
2.42
19.08
0.36
1.19
3.32
7.22
12.64
0.23
1.26
1.93
11.75
2.35
6.26
18.64
2.78
2.01
1.22
Figure 2: Synergy Score heat map of selected compounds screened in combination with eribulin and clustered based on target or pathway specificity. Twenty five cell lines were screened, shown as vertical columns labeled across the top and grouped first based on eribulin sensitivity/insensitivity then further clustered based on tumor type (breast cancer, blue; lung cancer, orange; ovarian cancer, green; single representative cancers, not highlighted; see Figure 1 for information on other cancer types). Each row lists one compound screened in combination with eribulin, with compounds clustered based on target or pathway specificity. Synergy Scores are shown, with higher scores highlighted in increasingly darker shades of red. For this analysis, a Synergy Score cut-off of 4.36 was chosen based on visual inspection of dose matrices and Loewe Excess matrices above and below this cut-off score.
75 126 147
50
80
94 131
.1
Eribulin(E7389) (uM)
18
7
29
22
4
13
23
-1
19
-11
9
5
-14
0
20 4
1.4e-4 4.1e-4 .0012 .0037
82
88
82
70
11
48
78
85
82
76
-3
33
48
76
71
86
5
25
34
58
73
94
19
21
42
56
73
.011
0
Eribulin(E7389) (uM)
15
10
11
-3
15
25
-7
2
-1
-12
6
74
68
71
78
-6
2
52
72
67
79
-15
-3
38
63
46
78
3
-15
-1
35
33
-6
2
-6
-8
93 135 160 142 174
73 0
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
74
94 130 129 146
-1
53
85
88 102
1.4e-4 4.1e-4 .0012 .0037
0
Eribulin(E7389) (uM)
.0012 .0037
.011 .033
8
Growth Inhibition (%) Vol=7.77(.5) Chi2=270
Lapatinib (uM)
9.9 3.3 1.1 .37 .12
-1
.1
.0012 .0037
.011 .033
0
39
41
53
59
54
0
43
43
34
17
-2
11
33
49
39
21
5
-3
20
23
31
11
16
5
15
13
24
-1
-4
0
.1
13
8
7
-4
.0012 .0037
.011 .033
4 .1
Eribulin(E7389) (uM)
Dose MatrixNone | 100 | 786-0 | ATP Lite | 72h |
DT-1308660
.011
-17
786-0 12
82
2
DT-1308669
51 6
1.4e-4 4.1e-4 .0012 .0037
.011
Eribulin(E7389) (uM)
Loewe ExcessNone | 100 | 7860 | ATP Lite | 72h |
DT-1308661
91 167 173 189 191 197 10
78
89 113 134 117
1
60
97 118 129 113
0
36
78 102 113 129
-2
25
44
76
94 126
-
27
32
42
71 112
0
.0012 .0037
.011 .033
.1
Eribulin(E7389) (uM)
N=2
49
12
Loewe ExcessNone | 100 | Hep G2 | ATP Lite | 72h |
Growth Inhibition (%)
26
-
Eribulin(E7389) (uM)
9.9
7
14
0
N=2 Growth Inhibition (%)
9.9 3.3 1.1 .37 .12 0
Lapatinib (uM)
Chi2=66 Growth Inhibition (%) Vol=4.7(.48)
9.9 3.3 1.1 .37 .12
-9
-6
DT-1308661
0
74
78
91
89
92
49
44
42
45
13
44
65
61
45
9
23
51
48
31
27
13
17
24
12
23
15
5
-10 -10
9
.011 .033
.1
0 1 0 -2 0
.0012 .0037
Eribulin(E7389) (uM)
A375 Dose MatrixNone | 100 | A375 | ATP Lite | 72h |
59
70
45
0
69
96
92
65
36
9
44
81
84
57
36
-4
1
39
65
30
19
-7
-6
-2
42
11
21
-7
-5
2
-1
0
.0012 .0037
.011 .033
1 .1
Eribulin(E7389) (uM)
66
86
97 135 142 166
Loewe ExcessNone | 100 | A375 | ATP Lite | 72h |
22
72
90 105 121 118
6
63
87 110 122 123
7
41
77
96 106 115
-3
38
52
75
96 119
20
30
53
74
0
1.4e-4 4.1e-4 .0012 .0037
96 .011
Eribulin(E7389) (uM)
DT-1308663
1
19
29
66
64
74
-1
33
38
40
43
26
5
43
50
51
44
31
7
25
43
39
29
23
-3
23
20
19
18
6
-1
-2
-4
9.9
41
3.3
37
1.1
0
DT-1308663
N=2
DT-1308662
0
1.4e-4 4.1e-4 .0012 .0037
27 5
Growth Inhibition (%) Vol=9.22(.44) Chi2=80
Loewe ExcessNone | 100 | T24 | ATP Lite | 72h |
Growth Inhibition (%)
Lapatinib (uM)
0
91
17
69
3.3
-1
9.9
63
22
91
3.3
13
.011 .033
41
88
1.1
-4
.0012 .0037
36
88
.37
-6
0
16
86
.12
76 111
13
7
1.1
1
Lapatinib (uM)
54
-6
53
98 149 168 164 175
0
-1
19
75 115 117 128 135 131
.37
22
Growth Inhibition (%) Vol=11.3(.55) Chi2=150
-6
38
12
90
12
6
Eribulin(E7389) (uM)
.12
64
28
8
DT-1308669
Lapatinib (uM)
53
9.9
.12
-7
58
109 149 183 185 184 187 90 109 170 165 167 174
36
6
0
0
7
3.3
94 109
29
Growth Inhibition (%) Vol=13.1(.41) Chi2=270
-9
1.1
79
11
9.9
-1
.37
40
-1
3.3
-1
.12
1
24
1.1
45
Lapatinib (uM)
.37
-4
44
.37
30
0
83 101 124 126
N=2
44
61
.12
-8
Eribulin(E7389) (uM)
Growth Inhibition (%)
1.1
9
9.9 3.3
73 107 127 135 126
DT-1308667
24
DT-1308660
38
1
Hep G2
Loewe ExcessNone | 100 | FaDu | ATP Lite | 72h |
T24
1
Eribulin(E7389) (uM) Dose MatrixNone | 100 | Hep G2 | ATP Lite | 72h |
Dose MatrixNone | 100 | FaDu | ATP Lite | 72h |
N=2
29
9.9
-41 -42 -24
9.9
N=2
27
DT-1308662
98 105 127 140 135
Eribulin(E7389) (uM)
12
Eribulin(E7389) (uM)
22
6
.1
Loewe ExcessNone | 100 | SNGM | ATP Lite | 72h |
2
.011
15
-4
Growth Inhibition (%) Vol=7.01(.44) Chi2=20
66
7
1.4e-4 4.1e-4 .0012 .0037
37
Growth Inhibition (%) Vol=11.1(.35) Chi2=190
32
0
64
9.9
5
51
3.3
83 136 150
37
1.1
63
14
-4
.37
14
DT-1308666
36
Dose MatrixNone | 100 | T24 | ATP Lite | 72h |
61
.011 .033
FaDu 34
Eribulin(E7389) (uM)
.0012 .0037
18
.12
20
.1
36
-
0
48
Lapatinib (uM)
64 113 148 149
Loewe ExcessNone | 100 | KYSE -410 | ATP Lite | 72h |
0
91 114
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
.37
97
55
53
0
42
Growth Inhibition (%)
78
49
65
9.9
16
6
3.3
80
38
36
0
3.3
8
86 135 145 146
1.1
79 105 115
37
-
18
1.1
-29 -14
57
.37
56
-3
99 120
31
.37
5
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
Lapatinib (uM)
9.9
N=2
0
79
53
.12
19
36
.12
28
0
-2
53
67
Lapatinib (uM)
-1
90 137 152 151
0
24
3
84 107 121 118
27
0
0
-4
74
Lapatinib (uM)
91 116 139
8
49
6
N=3-4
-22
Growth Inhibition (%) Vol=4.13(.49) Chi2=99
57
-10
-4
Loewe ExcessNone | 100 | NCIH460 | ATP Lite | 72h |
DT-1308664
Growth Inhibition (%)
6
3.3
37
6
98 135 130 121
9.9
12
1.1
.37
48
-
65
3.3
23
.37
96 143 154
-2
-4
1.1
21
2
.12
74
7
9
94 100 119 111 120
.37
1
Lapatinib (uM)
69
7
-2
5
.12
-7
-15 -27
0
1.1
62
14
0
DT-1308667
62
Eribulin(E7389) (uM)
Growth Inhibition (%)
9.9 3.3
91 107 145 160
.12
Lapatinib (uM)
73
0
74
11
13
96 100 103 111 142
0
-4
-2
24
22
6
29
Lapatinib (uM)
-1
2
DT-1308666
17
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
KYSE-410 181 186 193 192 193 185
7
Growth Inhibition (%) Vol=5.87(.48) Chi2=37
16
Dose MatrixNone | 100 | KYSE410 | ATP Lite | 72h |
10
9.9
22
Eribulin(E7389) (uM)
12
3.3
-1
.011 .033
23
1.1
-15
.0012 .0037
16
.37
-9
-
-2
.12
8
0
14
Lapatinib (uM)
-13
9.9 3.3
N=2
54
.1
19
.12
.011 .033
43
1.1
90 108 123 144 153 .0012 .0037
40
.37
0
38
.12
93 115 132 136 156
65
Lapatinib (uM)
13
30
DT-1308664
SNG-M
8
0
.37
92 121 110 139 160
25
Dose MatrixNone | 100 | SNG-M | ATP Lite | 72h |
DT-1308670
Growth Inhibition (%)
9.9 3.3 1.1
93 133 130 138 158
3
.12
Lapatinib (uM)
25
0
98 127 122 136 159
-1
Eribulin(E7389) (uM)
Loewe ExcessNone | 100 | A2780 | ATP Lite | 72h |
DT-1308670
30
0
Eribulin(E7389) (uM)
A2780
Dose MatrixNone | 100 | A2780 | ATP Lite | 72h |
86 154 174 176 180 191
.1
90 150 164 165
-19
Lapatinib (uM)
Eribulin(E7389) (uM)
.011 .033
.0012 .0037
54
29
0
0
-
34
0
.1
65 102 140 169 165
50
0
18
80 113 156 167 174
10
Growth Inhibition (%)
-3
3.3
-21 -16
1.1
6
.37
-
31
.12
23
0
N=2
9.9
17
-7
64
Lapatinib (uM)
.011 .033
3
-22 -14
59
0
.0012 .0037
-20 -20
-1
11
NCI-H460
Dose MatrixNone | 100 | NCIH460 | ATP Lite | 72h |
DT-1308665
N=2
0
-7
-4
86 128 154 170 174
9.9
93 115
19
34
3.3
81
21
91 139 172 179 181
1.1
76
5
22 -19 -26 -15
56
.37
69
54
.12
-
31
0
89 120
18
120 173 187 192 194 196
Lapatinib (uM)
82
-5
Loewe ExcessNone | 100 | MDAMB-468 | ATP Lite | 72h |
DT-1308665
Lapatinib (uM)
77 100 114
75
-9
-3
Growth Inhibition (%) Vol=2.46(.26) Chi2=330
77
68
65
Growth Inhibition (%) Vol=2.33(.51) Chi2=6.1
75
-4
59
3.3
.37
19
52
1.1
82 102 118
32
.37
71
27
.12
73
Lapatinib (uM)
1.1
24
4
0
92 114 127 151
Dose MatrixNone | 100 | MDAMB-468 | ATP Lite | 72h |
DT-1308668
Growth Inhibition (%)
9.9 3.3
88
.12
Lapatinib (uM)
46
0
132 156 161 181 188 194
MDA-MB-468
Loewe ExcessNone | 500 | MCF7 | ATP Lite | 72h |
DT-1308668
Growth Inhibition (%)
MCF7
Dose MatrixNone | 500 | MCF7 | ATP Lite | 72h |
.011
Eribulin(E7389) (uM)
Figure 3: Representative dose matrices for eribulin x lapatinib combination activity. Selected dose matrix pairs for eribulin in combination with lapatinib are shown in cell lines MCF7, MDA-MB-468, NCI-H460, A2780, SNG-M, Hep G2, KYSE-410, FaDu, 786-0, T24 and A375. For each cell line, Growth Inhibition values (left matrix) and Loewe Excess values (right matrix) are shown. The Growth Inhibition dose matrix values are determined by measurement of ATP levels with a T0 measurement (at time of drug addition) performed at the whole well level in order to distinguish cytostasis from cytotoxicity. Values highlighted in medium red are those approximating GI100% and are indicative of compounds/combinations being cytostatic; values with highlighting closest to black are those approximating GI200% and indicate complete killing (cytotoxic effect).
BKM-120, a pan-PI3K inhibitor. As shown in Figures 2, 4, one or more of these inhibitors are synergistic in combination with eribulin in the majority of cell lines in the panel. For a subset of cell lines, synergy is observed with all four PI3K pathway inhibitors. The breadth of activity observed in the cell line panel and for the various PI3K pathway inhibitors highlight the importance of PI3K signaling for tumor cell proliferation and/or survival upon eribulin exposure
012
(Figure 4). Combination activity is selective (for example, little or no combination activity in SK-BR-3, NCI-H552, NCI-H526, Mia PaCa2, and 786-0) pointing to specific genetic determinants in responsive cell lines as being important for combination activity.
MEK inhibitors Two MEK inhibitors (E6201 and trametinib) were screened in
Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identification of Combinatorial Drugs that Synergistically Kill both EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.
Rickles, et al. (2015)
HT-1080
T47D
NCI-H460
Hep G2
1.39
2.96
8.19
17.12
7.62
5.44
9.68
9.38
1.53
8.79
10.47
6.44
4.14
7.65
10.15
1.70
2.85
4.48
5.37
5.92
2.86
2.34
7.90
10.80
4.43
12.44
9.16
18.70
2.36
8.18
9.60
4.34
7.28
13.32 12.30 10.10
4.16
6.75
8.36
BKM-120
6.12
2.87
6.05
1.13
5.38
2.82
1.20
3.71
5.47
10.76
3.27
7.03
4.86
7.72
0.75
3.02
8.11
2.79
2.10
7.17
5.99
3.22
2.49
2.34
1.45
Everolimus 4.69
2.78
11.76
2.56
1.56
5.84
1.50
4.05
7.14
6.97
4.84
4.14
5.08
5.71
1.02
4.24
9.81
3.92
2.88
7.07
1.97
1.85
1.60
1.56
4.42
Dose Matrix | None | 100 | HT1080 | ATP Lite | 72h
103 153 173 185 186 186
139 148 165 163 170 180
.012 .037 .11
103 155 164 167 176 175
95 128 130 167 175 188
58 105 131 134 156 168
0
.011
.0012 .0037
.011 .033
.1
0
91 104
1.4e-4 4.1e-4 .0012 .0037
40
72 105 157 181
-
22
57
.011
0
96 151 180
Eribulin(E7389) (uM)
Eribulin(E7389) (uM)
Eribulin(E7389) (uM)
Eribulin(E7389) (uM)
Dose Matrix | None | 100 | MDA -MB-468 | ATP Lite | 72h
Dose Matrix | None | 500 | NCIH69 | ATP Lite | 72h
Dose Matrix | None | 100 | A2780 | ATP Lite | 72h
Dose Matrix | None | 100 | FaDu | ATP Lite | 72h
Dose Matrix | None | 100 | HT1080 | ATP Lite | 72h
104 121 156 165 168 164
164 170 171 171 175 174
122 161 169 181 177 185
84 106 142 141 158 164
0
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
Eribulin(E7389) (uM)
0
1.4e-4 4.1e-4 .0012 .0037
.011
Eribulin(E7389) (uM)
54
89
92
Dose MatrixNone | 100 | FaDu | ATP Lite | 72h |
74 111 147 151 169 179
44
58
73 101 132 136
42
59
75
95 108 147
34
43
72
89
37
57
68
89 103 128
33
35
77
92
93 128
-
-1
53
85
88 102
30
97 136 136 159 173
14
90 123 123 143 161
4
90 111 130 147 159
-
90 108 123 144 153
0
.0012 .0037
.011 .033
92 150 181
2
96 141 178
66
-5 0
.011
Dose MatrixNone | 100 | A2780 | ATP Lite | 72h |
DT-1308899
N=1-2
99
.66
6
1.4e-4 4.1e-4 .0012 .0037
.025 .074 .22
91 111
70
30
0
81
35
BKM-120 (uM)
60
Eribulin(E7389) (uM)
62 110 148 141 163 180
Growth Inhibition (%)
2 .66 .025 .074 .22
BKM-120 (uM)
0
N=2
2 .66 .025 .074 .22
BKM-120 (uM)
Growth Inhibition (%)
0
44
8 12
.1
Eribulin(E7389) (uM)
0
1.4e-4 4.1e-4 .0012 .0037
.011
Eribulin(E7389) (uM)
63
92 145 175
25
56
89 124 163
82 112 144 177 185
54
73
50
70 104 145 173 184
53
74
96 153 171 182
-
43
54 122 150 168
0
97 144 169 180
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
Eribulin(E7389) (uM)
99 102 90
.0012 .0037
.011 .033
67
Growth Inhibition (%)
.7 .026 .078 .23 .0086
N=2
BEZ235
.1
DT-1308718
94 129 127 141 128 132 92 106 127 120 104
19
79
13
57 108 113 107 67
13
63
98 106 103 74
63
90 101 98
0
99 123 104 83
.0012 .0037
.011 .033
72
BKM-120
.1
Eribulin(E7389) (uM) Dose MatrixNone | 100 | T47D | ATP Lite | 72h |
DT-1308900
76 107 147 175 175
60
32
Dose MatrixNone | 100 | HT1080 | ATP Lite | 72h |
48
83 100 115 118 95
-
Dose Matrix | None | 100 | T47D | ATP Lite | 72h
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
51
55
0
Eribulin(E7389) (uM)
DT-1308895
98 142
26
.017 .05
66 121 133 135
.1
-3
99 114
77 119 174 180
6.2e-4 .0019 .0055
12
84
59
Everolimus (uM)
-
86 100 128
62
99 145 161 171 186
19
97 103 125 140 142 132
Eribulin(E7389) (uM)
DT-1308722
85
0
65 134 150 145
75
37
N=2
72 134 151 155
21
43
Growth Inhibition (%)
76 139 146 160
26
14
9 18
.017 .05
33
14
.011 .033
95 113 162
6.2e-4 .0019 .0055
20
90 100 109 129 143 .0012 .0037
84
Eribulin(E7389) (uM)
N=2
N=2
86 151 163 164
91 106 110 130 149
66
Everolimus (uM)
90 150 164 165
53
-
94 114 123 138 159
23
0
54
56 106 150 162 166
23
7.6e-6 2.3e-5 6.8e-5 2.1e-4 6.2e-4
-
7.6e-6 2.3e-5 6.8e-5 2.1e-4 6.2e-4
91 154 171 168
Everolimus (uM)
81
0
72 127 157 172 177
7
Growth Inhibition (%)
7.6e-6 2.3e-5 6.8e-5 2.1e-4 6.2e-4
0
84 130 163 185 186
2
DT-1308896
35
2
0
.011
Dose MatrixNone | 500 | NCIH69 | ATP Lite | 72h |
DT-1308898
25
62 126 143 147
N=2
2
Dose MatrixNone | 100 | MDAMB-468 | ATP Lite | 72h |
39 117 162 176 185 183
27
1.4e-4 4.1e-4 .0012 .0037
Eribulin(E7389) (uM)
Eribulin(E7389) (uM)
64 119 158 185 187 187
62 136 135 153
21
DT-1308721
Growth Inhibition (%)
0
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
20
0
1
30 109 130 132 151 166
Everolimus (uM)
64 106 136 142
76 133 151 156
75 125 148 147 160 179
0
45
98 148 158 160
41
Growth Inhibition (%)
63 110 137 142
58
16
N=2
15
34
DT-1308720
Growth Inhibition (%)
0
97 143 132
.66
7
59
93 140 160 167 168
.025 .074 .22
72 148 146 155
74
0
16
N=2-3
13
-22 29
DT-1308719
BKM-120 (uM)
99 142 150 163
Growth Inhibition (%)
0
.025 .074 .22
.66
BKM-120 (uM)
2
DT-1308717
54
107 120 134 150 156 145
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
Eribulin(E7389) (uM)
6
1
18
.33
85 112 173 187
BEZ235 (uM)
62
.012 .037 .11
99 104 153 175 184
30
0
84
.012 .037 .11
52
.0014 .0041
-4
77
0
-
N=1-2
66 105 97 153
BEZ235 (uM)
33
Growth Inhibition (%)
N=2
24
98 140 160 161
104 123 141 146 150 141
2
1.4e-4 4.1e-4 .0012 .0037
89 108 115 133 159
73 105 102 142 172
DT-1308725
100 125 139 138 145 147
.66
0
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
-
1
58 126 144 146
94 134 127 139 165
.33
15
33
72
49
BEZ235 (uM)
-
N=2
N=1-2
.012 .037 .11
83 140 147 159
67
82 115 126 163 179 188
.1
.025 .074 .22
0
33
.012 .037 .11
82 120 143 158
16
89 109 143 147 166 180
81 102 125 152 170 176
66
Dose Matrix | None | 100 | T47D | ATP Lite | 72h
DT-1308728
DT-1308723
100 123 159 170 178 170
0
61
85 147 158 163
99 138 159 163 173 180
Growth Inhibition (%)
-
32
BEZ235 (uM)
88 134 168 165
52 112 155 166 165
13
0
33
57
DT-1308727
Growth Inhibition (%)
25
.0014 .0041
66 105 146 173 176
BEZ235 (uM)
22
103 113 148 164 169 174
0
0
.0014 .0041
48 103 149 173 182 182
DT-1308726
Growth Inhibition (%)
93 127 173 184 191 187
.011 .033
N=1-2
Dose Matrix | None | 100 | FaDu | ATP Lite | 72h
71 100 100 94 .0012 .0037
AZD 8055
Eribulin(E7389) (uM)
Growth Inhibition (%)
Dose Matrix | None | 100 | A2780 | ATP Lite | 72h
-
96 109 106 79
N=2
Dose Matrix | None | 500 | NCIH69 | ATP Lite | 72h
76
Growth Inhibition (%)
Dose Matrix | None | 100 | MDA -MB-468 | ATP Lite | 72h
80 103 112 106 92
35
DT-1308897
32
84 106 129 106 102
31
74 105 111 118 96
15
74 106 120 118 81
36
75 108 117 114 90
29
83 112 111 104 90
-
51
0
88 104 94
.0012 .0037
.011 .033
73
N=1-2
Eribulin(E7389) (uM)
56
0
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
Eribulin(E7389) (uM)
81 104 116 113 128 111
Growth Inhibition (%)
86 135 174
AZD 8055 (uM)
64
0
0
14
.011
Eribulin(E7389) (uM)
BKM-120 (uM)
95 102
74 102 146 179
0
89
1.4e-4 4.1e-4 .0012 .0037
38
99 109 130 132 129 125
.017 .05
54
0
11
100 128 126 141 141 127
6.2e-4 .0019 .0055
3
.1
83 109 171 184
Everolimus (uM)
94 134
92 140 174 187
52
0
80
70
32
N=2
56
57
Growth Inhibition (%)
25
.7 .026 .078 .23
1
AZD 8055 (uM)
N=2
97 129
.0014 .0041
.011 .033
94
.0086
91 104 108 128 147 .0012 .0037
58
0
0
.011
Growth Inhibition (%)
.7 .026 .078 .23 .0086
AZD 8055 (uM)
0
N=2 Growth Inhibition (%)
.7 .026 .078 .23 .0086
0
N=2-3
AZD 8055 (uM)
Growth Inhibition (%)
AZD 8055 (uM)
0
61 130 140 150
1.4e-4 4.1e-4 .0012 .0037
74 103 130 166
15
Eribulin(E7389) (uM)
BEZ235 (uM)
.012 .037 .11
21
0
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
54 110 138 143 152 177
39
10
Eribulin(E7389) (uM) DT-1308724
Everolimus (uM)
87 147 161 162
25
N=2
64 106 136 150
46
76 110 134 152 170 179
DT-1308695
Growth Inhibition (%)
52
0
19
90 126 150 157 173 182
91 110 147 176 185
N=2
69 107 135 154
62 100 153 163 164
77
Growth Inhibition (%)
38
84 123 159 165 168
39
87 117 142 155 179 184
N=2
3
57
94 106 145 162
Growth Inhibition (%)
60 109 152 142
66
.7
71 132 159 162
26
73 106 146 144 178
56
.026 .078 .23
55
14
73
.0086
17
97 137 158 167 177 184
DT-1308706
0
103 152 166 170 177 178
104 104 143 163 162 166
N=1-2
132 132 149 159 164 169
62 100 157 180 174
T47D Dose Matrix | None | 100 | T47D | ATP Lite | 72h
Dose Matrix | None | 100 | HT1080 | ATP Lite | 72h
DT-1308703
AZD 8055 (uM)
61 121 174 184 179
26
HT-1080
Dose Matrix | None | 100 | FaDu | ATP Lite | 72h
DT-1308693
Growth Inhibition (%)
47
DT-1308699
FaDu
FaDu
Dose Matrix | None | 100 | A2780 | ATP Lite | 72h
DT-1308694
SNG-M
A2780
A2780
Dose Matrix | None | 500 | NCIH69 | ATP Lite | 72h
.026 .078
NCI-H69
Dose Matrix | None | 100 | MDA -MB-468 | ATP Lite | 72h
9.6e-4 .0029 .0086
MDA-MB-468
A375
PC-3
6.53
6.78
T24
NCI-H526
7.71
1.32
786-0
NCI-H522
1.13
16.92
KATOIII
NCI-H1650
8.60
5.32
MIA PaCa-2
A549
5.04
8.03
KYSE-410
SK-BR-3
11.84
BEZ235
SK-OV-3
MDA-MB-468
AZD 8055
NCI-H69
MDA-MB-436
PI3K Pathway
Eribulin +
MCF7
Target
Eribulin-Insensitve Cell Lines MDA-MB-231
Eribulin-Sensitive Cell Lines
Everolimus
.1
Eribulin(E7389) (uM)
Figure 4: Synergy Score heat map and representative Growth Inhibition dose matrices for eribulin x PI3K pathway combination activities. Select pairs of dose matrix plots for eribulin in combination with PI3K pathway inhibitors are shown for cell lines MDA-MB-468, NCI-H69, A2780, HT-1080 and T47D. Growth Inhibition dose matrices for AZD8055 (mTOR TORC1/2 kinase inhibitor), BEZ235 (pan-PI3K/mTOR inhibitor), BKM-120 (pan-PI3K inhibitor) and everolimus (mTOR TORC1 inhibitor) are shown.
combination with eribulin (Figure 2); selected Growth Inhibition and Loewe Excess dose matrices are shown in Figure 5. There is good concordance for both inhibitors; when synergy is observed with one of the MEK inhibitors, it is most often observed with the other. MEK combination activity was strongest in MDA-MB-231, A2780 and Hep G2. For some cell lines, synergy is observed when eribulin is combined with either PI3K pathway inhibitors or MEK inhibitors (for example, FaDu, NCI-H460 and A2780). For other cell lines, (MCF7, NCI-H69, KYSE-410, HT-1080 and T47D), synergies are PI3K pathway-specific, with no synergies seen for MEK inhibitors.
BCL-2 Inhibition ABT-263 (Navitoclax) is a potent Bcl-xL, Bcl-2, and Bcl-w inhibitor currently in multiple clinical trials as a monotherapy or in combination with approved drugs. Strong synergies with eribulin and good breadth of activity was observed with ABT-263, in both eribulin-sensitive and insensitive cell lines (Figure 6). Strong synergy was observed in most but not all of the cell lines, suggesting that an
013
eribulin x ABT-263 combination is not non-selectively synergistically toxic.
Antagonistic drug pairings As described in Materials and Methods, Loewe Volume values can be used to gauge both synergies and antagonisms. A Loewe Volume heat map is shown in Figure 7 for 17-DMAG, cytarabine, topotecan, and gemcitabine, with greater negative values (antagonism) highlighted in increasingly darker shades of blue and increasing positive values (synergies) highlighted in increasingly darker shades of red. Also shown are representative combination dose matrices for selected antagonistic activities observed. The 17-DMAG Loewe Volume combination activity pattern is complex: for 6 cell lines (red shaded), combinations with eribulin are synergistic, while for 4 cell lines, antagonism was observed.
Discussion Eribulin has broad anticancer activity in a wide variety of
Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identification of Combinatorial Drugs that Synergistically Kill both EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.
Rickles, et al. (2015)
-6 -13 -10 -1
7
46
47
69
86
.0012 .0037
.1
5
10 3.3 1.1 .37 .12
ANDI(E6201) (uM)
-1
0
-5
.011 .033
.1
0
91 104 108 128 147 .0012 .0037
.011 .033
DT-1308808
Dose MatrixNone | 100 | A2780 | ATP Lite | 72h |
3
27
57
57
64
57
-2
18
30
27
36
45
-5
12
22
20
24
34
.025 .074 .22
95 139 156 157 167 176
.66
2
8
21
20
8
18
67 108 135 140 157 172
0
7
14
11
7
22
.0027 .0082
Loewe ExcessNone | 100 | NCIH460 | ATP Lite | 72h |
71
84
94
95 112
48
69
77
85
93 116
46
47
69
86
0
.0012 .0037
99
.011 .033
11
-8
-5
-1
.1
0
Eribulin(E7389) (uM)
.0012 .0037
5
.011 .033
Trametinib (uM)
65
0
93 111 128
10
9
12
38
41
-24 -19
0
19
-
6
0
.1
89 127 151 156 161 173 84 112 141 145 158 171
48 109 131 133 147 162 0
Eribulin(E7389) (uM)
89 100 101 118 149 .0012 .0037
.011 .033
.0012 .0037
.011 .033
33
30
73
73
-1
8
14
28
48
61
6
-8
7
-7
-
.1
0
75
81
16
14
.0012 .0037
94 116 130 46
47
66
.011 .033
.1
0
.0012 .0037
.011 .033
2
Eribulin(E7389) (uM)
Loewe ExcessNone | 100 | A2780 | ATP Lite | 72h |
Dose MatrixNone | 100 | Hep G2 | ATP Lite | 72h |
Loewe ExcessNone | 100 | Hep G2 | ATP Lite | 72h |
0
44
59
41
34
33
-2
35
55
39
29
30
1
24
45
29
26
28
-1
27
41
24
24
29
0
35
37
17
14
19
20
6
-
.0012 .0037
-15 -15
6
.011 .033
.1
DT-1308810
157 167 174 174 178 188 139 147 160 158 173 181 89 110 114 142 134 158 56
65
77
87
98 131
46
57
57
65
67
98
9
16
37
50
77
0
Eribulin(E7389) (uM)
.0012 .0037
.011 .033
E6201
.1
Eribulin(E7389) (uM)
0
Eribulin(E7389) (uM)
27
65
DT-1308809
.1
62
4
99 122 128 125 168 168
Eribulin(E7389) (uM)
DT-1308809
Growth Inhibition (%) Vol=6.67(.45) Chi2=53
85
2
75
N=2
.025 .074 .22
58
Trametinib (uM)
94 101 122 139
0
81
Growth Inhibition (%)
.66
61
6
.1
Dose MatrixNone | 100 | NCIH460 | ATP Lite | 72h |
DT-1308808
2
-8
Eribulin(E7389) (uM)
91 121 131 151 151
Trametinib (uM)
11
.0012 .0037
Eribulin(E7389) (uM)
66
0
0
Growth Inhibition (%) Vol=4.31(.43) Chi2=15
N=1-2
ANDI(E6201) (uM)
0
Growth Inhibition (%)
99
.011 .033
Eribulin(E7389) (uM)
.025 .074 .22
ANDI(E6201) (uM)
0
0
37 119 136 139 165 169
50
Growth Inhibition (%) Vol=9.53(.86) Chi2=40
.12
-4
39
DT-1308810
.1
5
15
22
22
26
36
6
13
27
24
40
47
-12
9
12
41
32
56
-6
4
13
20
24
50
15
23
19
16
4
22
3
0
3
-7
0
Eribulin(E7389) (uM)
.0012 .0037
.011 .033
4
Growth Inhibition (%) Vol=6.16(.49) Chi2=17
86 101
27
10
64
22
N=2
44
16
3.3
33
5
1.1
-1
70 139 150 155 166 177
.37
27
61
.12
10
56
ANDI(E6201) (uM)
5
41
0
-2
41
Growth Inhibition (%)
9
42
-3
121 165 164 179 185 186
9.1e-4 .0027 .0082
.37
1
56
1e-4 3e-4
97 121
50
0
79
47
N=1-2
58
38
Trametinib (uM)
55
-13 35
DT-1308818
Growth Inhibition (%)
19
94 162 165 174 177 184
27
37
10
22
21
37
3.3
12
22
34
1.1
15
21
30
.37
21
21
-3
143 177 181 183 184 188
.12
16
50
ANDI(E6201) (uM)
1.1
0
50
0
98 117
48
9.1e-4 .0027 .0082
89
46
1e-4 3e-4
87
46
Trametinib (uM)
76
-3
Loewe Excess | None | 100 | Hep G2 | ATP Lite | 72h
2
164 183 183 184 184 190
0
1.1
55
138 187 187 189 191 192
Growth Inhibition (%) Vol=8.91(.19) Chi2=580
38
Dose Matrix | None | 100 | Hep G2 | ATP Lite | 72h
DT-1308818
Growth Inhibition (%) Vol=8.17(.48) Chi2=67
42
10
37
34
3.3
21
35
1.1
7
31
.37
3.3
-4
32
.12
90 111 128 132
30
0
74
11
.025 .074 .22
3.3
63
171 190 192 191 195 194
.0027 .0082
50
0
42
N=2
34
ANDI(E6201) (uM)
36
DT-1308813
Growth Inhibition (%)
14
Loewe Excess | None | 100 | A2780 | ATP Lite | 72h
DT-1308813
N=2
10
2
Hep G2
Dose Matrix | None | 100 | A2780 | ATP Lite | 72h
DT-1308817
Trametinib (uM)
82 105 108 128 144
DT-1308817
Growth Inhibition (%)
10
71
.37
A2780
Loewe Excess | None | 100 | NCI-H460 | ATP Lite | 72h
.12
NCI-H460 Dose Matrix | None | 100 | NCIH460 | ATP Lite | 72h
Trametinib
.1
Eribulin(E7389) (uM)
Figure 5: Eribulin x MEK inhibitor combination activities. Select dose matrices (Growth Inhibition and Loewe Excess) for eribulin in combination with the MEK inhibitors E6201 and trametinib are shown for cell lines NCI-H460, A2780 and Hep G2. Breadth of combination activity is shown in Figure 2 (Synergy Score heat map).
KATOIII
MDA-MB-231
T47D
NCI-H460
Hep G2
786-0
T24
A375
9.21
12.9
5.24
13.6
13
12.2
14.9
8.76
8.53
22
15.1
6.99
13.1
9.49
14.5
11.8
0
.0012 .0037
.011 .033
.1
Eribulin(E7389) (uM)
1
-
42
68
.0012 .0037
74
75
.011 .033
5
-21 -11
0
14
0
.0012 .0037
.011 .033
.1
Eribulin(E7389) (uM)
36 100 140 146 149
-
17
0
85 128 138 148
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
Eribulin(E7389) (uM)
6
4
6
-1
3
39
9
4
6
6
-5
35
6
4
4
12
6
15
8
3
5
-
-3
3
-4
-5
4
0
0
0
0
0
.011 .033
10
Chi2=180
.1
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
Eribulin(E7389) (uM)
41
63
78
86 160 166
4
27
72
81 129 150
-6
24
54
82 103 153
12
13
42
76 104 125
19
11
64
78
94 125
-
9
42
65
91 101
0
1.4e-4 4.1e-4 .0012 .0037
.011
Eribulin(E7389) (uM)
Loewe Excess | None | 100 | SNG-M | ATP Lite | 72h
0
21
36
18
69
66
0
-8
31
13
38
50
-6
1
13
14
12
52
12
-5
1
8
13
24
19
-4
25
10
3
24
-
-5
5
-3
0
0
1.4e-4 4.1e-4 .0012 .0037
0
Chi2=8
-
13
0
.0012 .0037
Growth Inhibition (%) Vol=5.24(.53)
27
0
Eribulin(E7389) (uM)
.31
16
31
.31
5
-9
.019 .039 .078 .16
-3
-7
0
-1
31 119 138 147 148
-9
ABT-263 (uM)
5
12
-
.1
Dose Matrix | None | 100 | SNGM | ATP Lite | 72h
Growth Inhibition (%) Vol=2.33(.36) Chi2=13
35
-9
10
28
7
5
16
37
2.5
13
27
1.2
5
55 123 141 147 151
6
.62
0
21
113 116 113 120 118
SNG-M
0
39
N=1-2
28
5
22 108 115 115 116 115
Eribulin(E7389) (uM)
Loewe Excess | None | 500 | SK -BR-3 | ATP Lite | 72h
ABT-263 (uM)
20
58 115 138 147 150
76
N=2
10
0
-18 98 100 101 99 102
Growth Inhibition (%) Vol=22(.36)
-1
5
-4
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
69
2.5
5
53 168 187 190 192 190
45
69
1.2
-7
0
62 184 190 189 196 194
41
65
.62
99
43
67
ABT-263 (uM)
98
25
0
36
Growth Inhibition (%)
69 102 101
28
Eribulin(E7389) (uM)
Growth Inhibition (%)
18
10
17
5
-1
42
2.5
34
1.2
34
10
N=2
31
.62
94 108
28
0
83
-11 19
ABT-263 (uM)
73
37
Chi2=29
68
35
Growth Inhibition (%) Vol=5.56(.3)
-
29
5
99 110 121
13
4
41
.019 .039 .078 .16
76 103 122 139 134 135
15
2.5
91
17
40
-32 65
ABT-263 (uM)
7
1.2
83
3 26
74 190 192 193 191 194
SK-BR-3
.62
25
58 100 103
95 192 193 192 196 196
0
Dose Matrix | None | 500 | SKBR-3 | ATP Lite | 72h
29
ABT-263 (uM)
94 107 110 122 129
95
Loewe Excess | None | 500 | MCF7 | ATP Lite | 72h
0
1.2
37
Growth Inhibition (%)
10 5 2.5
57 109 112 114 122 133
.62
ABT-263 (uM)
69 113 122 125 128 128
0
102 124 110 125 131 132
91
15
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
MCF7 Dose Matrix | None | 500 | MCF7 | ATP Lite | 72h
73
-3
5
41
98
2.5
1
0
Eribulin(E7389) (uM)
98
Loewe Excess | None | 100 | MDA-MB-231 | ATP Lite | 72h
N=2
-1 .011
Eribulin(E7389) (uM)
-25 -36 -13 73
178 194 195 197 195 199
1.2
3
74 117 190 194
71
.62
1
1.4e-4 4.1e-4 .0012 .0037
67 151 194 195
22
71
ABT-263 (uM)
-3
0
46
29
69
0
5
.011
15
10
86
5
89
90
48
9
2.5
96
58
39
1.2
74
35
85 146 194 198
13
.62
27
32
52
0
34
47
N=2
-5 17
80 167 193 193
ABT-263 (uM)
95
53
MDA-MB-231 Dose Matrix | None | 100 | MDA -MB-231 | ATP Lite | 72h
Loewe Excess | None | 100 | KYSE-410 | ATP Lite | 72h
Growth Inhibition (%)
92
10
70
5
14
60
2.5
20
ABT-263 (uM)
11
10
N=2
64
1.2
84
58
140 165 175 196 198 197
.62
82
55
Eribulin(E7389) (uM)
12
0
61
14
Dose Matrix | None | 100 | KYSE -410 | ATP Lite | 72h
Growth Inhibition (%) Vol=11.8(.72) Chi2=110
24
1.4e-4 4.1e-4 .0012 .0037
5
12
0
-23 -13 13
2.5
75 125 171 171
16
1.2
79 146 179 174
50
24
.62
66
22
20
ABT-263 (uM)
15
16
0
90 152 177 181
Growth Inhibition (%)
10 2.5
85
0
70
1.2
100 109 135 177 180 187
.62
5
ABT-263 (uM)
180 180 183 187 191 183
KYSE-410
Loewe Excess | None | 100 | NCI-H1650 | ATP Lite | 72h
Growth Inhibition (%) Vol=13.6(.49) Chi2=1200
NCI-H1650 Dose Matrix | None | 100 | NCIH1650 | ATP Lite | 72h
Growth Inhibition (%)
0.928 0.663
HT-1080
2.59
PC-3
11.8
MIA PaCa-2
NCI-H522
12.6
FaDu
NCI-H1650
2.33
KYSE-410
A549
12.1
SNG-M
SK-BR-3
5.77
SK-OV-3
MDA-MB-468
5.56
Eribulin-Insensitve Cell Lines
A2780
MDA-MB-436
ABT-263
NCI-H69
Eribulin +
BCL2
NCI-H526
Target
MCF7
Eribulin-Sensitive Cell Lines
.011
Eribulin(E7389) (uM)
Figure 6: Synergy Score heat map and representative Growth Inhibition dose matrices for eribulin x ABT-263. Select pairs of dose matrix plots for eribulin in combination with the BCL-2 family inhibitor ABT-263 are shown for cell lines NCI-H1650, KYSE-410, MDA-MB-231, MCF7, SK-BR-3 and SNG-M. Three strong synergies are highlighted (black arrows, top row of matrices) as well as cell lines with weak combination effects (grey arrows, bottom row of matrices).
preclinical cancer models [4,7] and numerous monotherapy clinical trials are ongoing to investigate efficacy in non-breast tumor types (see www.clinicaltrials.gov). Consistent with utility for additional potential indications, a variety of tumor cell lines including lung, ovarian, endometrial, head and neck, prostate and melanoma are shown here to be sensitive to eribulin at clinically relevant concentrations. Since tumor cells have a robust capacity to lessen the therapeutic effects of cancer drugs through adaptive and acquired resistance mechanisms, the identification of drugs that can
014
paired with eribulin to trigger selective and synergistic tumor cell death represents a critical path toward optimizing patient benefit. Specifically, drug combinations need to be identified for situations in which the effects of eribulin monotherapy are suboptimal, or in which intrinsic or acquired drug resistance might be overcome with strategically selected drug combinations. Not only eribulin, but also all anticancer drugs need to be looked for appropriate combination partners. A molecular targeted agent,
Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identification of Combinatorial Drugs that Synergistically Kill both EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.
Rickles, et al. (2015)
Eribulin-Insensitve Cell Lines
2.01
-0.56
1.18
1.83
0.94
-1.44
1.47
1.10
0.09
-0.93
-1.52
-1.94
3.05
3.44
1.94
2.11
0.74
1.05
1.22
-2.97
-8.20
-0.48
-4.03
-5.42
-2.15
-2.43
-6.28
1.09
-2.28
-5.81
-5.53
-10.65
-2.28
-7.96
0.32
-0.05
-0.69
0.35
-1.21
Topotecan
-1.48
-0.59
-3.14
-4.16
-0.17
-1.87
-5.33
-1.87
-3.47
-4.55
-1.79
-3.49
-5.15
1.19
-1.76
-1.68
-4.50
-11.00
-0.39
-2.89
-0.17
1.14
0.31
-0.88
-0.68
Gemcitabine
-4.28
-3.41
-4.49
-4.48
-0.02
-0.88
-5.23
-0.11
-1.72
-2.30
-0.24
-1.85
-3.07
0.65
-2.00
-2.19
-2.99
-13.86
-0.73
-8.03
-0.23
0.86
1.09
-1.43
-2.55
-7
11
25
62
89 108
.37
23
17
44
73
82
70
-9
9
29
56 113 104
.12
5
32
47
86 108 122
7
43
74 115 120
53
64 106 136 150
40
92 144 152 155
0
Eribulin(E7389) (uM)
82
80
61
82
84
0
.011
Eribulin(E7389) (uM)
92
90 102 93 106 118
73
67
66
82
56
51
90 100 100
20
63 121 130 130
53 0
91
1.4e-4 4.1e-4 .0012 .0037
98
79
87
99
20
42
75 110 156
-8
5
35
82 139 180
22
57
96 151 180
0
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
18
10
13
12
5
1
8
-4
-10
-3
-29 -28
-8
-22 -11
-44
4
20
7
20
25
-32
6
29
36
46
53
35
47
52
56
0
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
Eribulin(E7389) (uM)
1.4e-4 4.1e-4 .0012 .0037
3
63 .011
KYSE-410
HT-1080
Dose MatrixNone | 100 | KYSE410 | ATP Lite | 72h |
Dose MatrixNone | 100 | HT1080 | ATP Lite | 72h |
96
96 102 97 104 118
118 111 109 111 108 120
92
97
74
93 101 112 103 113
26
91 111 121 126 145
.011
Eribulin(E7389) (uM)
7 0
97 105 102 107
89 110 117 125 152 89 108 115 133 159 .0012 .0037
.011 .033
.1
Eribulin(E7389) (uM)
1.1
DT-1308945
76
73
72
70
75
78
48
39
46
44
55
50
25
26
28
40
37
53
-5
27
64
89
92
-5
43
79
87
99
5 0
Cytarabine
Eribulin(E7389) (uM)
Dose MatrixNone | 100 | A2780 | ATP Lite | 72h |
DT-1308942
T24
786-0
Hep G2
T47D
KATOIII
PC-3
FaDu
SNG-M
43
9.9
N=2
-5
0
14
Eribulin(E7389) (uM)
N=2
N=1-2
N=2-3
65
87 116
.37
51
24
1.4e-4 4.1e-4 .0012 .0037
54
76
.014 .041 .12
37
12
-
31
63
0
16
23
15
49
Topotecan Hydrochloride (uM)
63
47
Growth Inhibition (%)
59
41
30
0
71
34
59
Topotecan Hydrochloride (uM)
76
27
.014 .041 .12
67
30
0
93 111 139
64 106 136 150
54
28
Topotecan Hydrochloride (uM)
68
53
54
Growth Inhibition (%)
65
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
.014 .041 .12
82
0
73
42
DT-1308947
DT-1308943
Topotecan Hydrochloride (uM)
-
69
Growth Inhibition (%)
1.1 .37 .014 .041 .12
31
71
49
52
DT-1308940
13 -16
.014 .041 .12
.37
123 119 122 136 127 136
63
43
95 111
.0015 .0046
1.1
109 103 124 115 111 117
53
35
91
A2780
146 143 141 149 148 154
113 130 109 113 113 105
44
NCI-H69 Dose MatrixNone | 500 | NCIH69 | ATP Lite | 72h |
1.1
NCI-H1650 Dose MatrixNone | 100 | NCIH1650 | ATP Lite | 72h |
160 160 156 164 167 172
50
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
162 154 157 153 154 158
DT-1308946
50
Eribulin(E7389) (uM)
.37
MDA-MB-468
0
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
Eribulin(E7389) (uM)
3.3
95 119 129 131
1.1
50
44
.37
35
44
.12
98 107 109
44
0
90
40
Cytarabine (uM)
80
Growth Inhibition (%)
1.1 .37 .014 .041 .12
Cytarabine (uM)
74
0
N=2-3
99 111 111
Dose MatrixNone | 100 | MDAMB-468 | ATP Lite | 72h |
176 177 181 183 183 179
0
98
N=2
0
Growth Inhibition (%)
9.9
Cytarabine (uM)
-
0
N=2
.011
91
Growth Inhibition (%)
1.4e-4 4.1e-4 .0012 .0037
Growth Inhibition (%)
1.1
Cytarabine (uM)
0
0
90
90
N=2
62
81
Growth Inhibition (%)
52
78
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
Eribulin(E7389) (uM)
DT-1308944
190 186 191 190 194 193 144 152 158 166 156 168 115 117 118 125 129 145 88 59 0
88
91
92
95 108
71
74
78
84
22
57
96 151 180
90
N=2
60
101 111 111 116 128 136
Growth Inhibition (%)
50
44
9.9
53
54
55
3.3
1.1
34
56
53
1.1
63
56
52
.37
63
44
46
.12
42
55
39
Cytarabine (uM)
28
51
0
3.3
24
89 102 98 106 113
9.9
.37 .014 .041 .12
13
96
3.3
98 101 94 105 119 125
78
1.1
91
64
.37
89
78
.12
86
58
0
77
62
N=2
86
47
Growth Inhibition (%)
69
63
N=2
64
63
Topotecan Hydrochloride (uM)
68
64
Growth Inhibition (%)
72
41
1.1
69
60
KATOIII Dose Matrix | None | 500 | KATOIII | ATP Lite | 72h
DT-1308937
.37
59
36
HT-1080 Dose Matrix | None | 100 | HT1080 | ATP Lite | 72h
DT-1308939
.014 .041 .12
69
Eribulin(E7389) (uM)
KYSE-410 Dose Matrix | None | 100 | KYSE -410 | ATP Lite | 72h
DT-1308938
Cytarabine (uM)
Dose Matrix | None | 500 | SKBR-3 | ATP Lite | 72h
0
DT-1308941
N=1-2
SK-BR-3
Dose Matrix | None | 100 | MDA -MB-468 | ATP Lite | 72h
Growth Inhibition (%)
MDA-MB-468
DT-1308936
Topotecan Hydrochloride (uM)
A2780
MDA-MB-436 Dose Matrix | None | 100 | MDA -MB-436 | ATP Lite | 72h
A375
0.84
-1.32
NCI-H460
NCI-H526
-2.30
-5.94
HT-1080
NCI-H522
-5.95
-8.73
MIA PaCa-2
A549
-5.07
-5.39
KYSE-410
SK-BR-3
0.10
-3.92
SK-OV-3
MDA-MB-468
-3.35
Cytarabine
NCI-H69
MDA-MB-436
17-DMAG
NCI-H1650
Eribulin +
MCF7
MDA-MB-231
Eribulin-Sensitive Cell Lines
Topotecan
4.6e-5 1.4e-4 4.1e-4 .0012 .0037
Eribulin(E7389) (uM)
Figure 7: Loewe Volume heat map of select compounds screened in combination with eribulin. Loewe Volume scores are shown with potential synergies in pink/red and potential antagonisms in aqua/blue. Also shown are Growth Inhibition dose matrices for eribulin x cytarabine and eribulin x topotecan in the cell lines MDA-MB-436, MDA-MB-468, SK-BR-3, KYSE-410, HT-1080 and Kato III.
vemurafenib (BRAF inhibitor), demonstrated 48% anti-tumor response and extended PFS to 5.3 months in V600E melanoma patients in the BRIM3 study [21], in a first-line BRAF mutated melanoma therapy. However, the treatment of vemurafenib caused resistance in some patients through its 2-18 months post treatment [22]. Recently, four articles reported five resistance mechanisms for vemurafenib [23-27]. Those authors attributed their resistance mechanisms to the overexpression of PDGFR-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 eribulinsensitive and insensitive cell lines, suggesting potential clinical benefit for both responders and non-responders of eribulin monotherapy. In addition to approved drugs that target EGFR/HER2 (erlotinib, lapatinib, BIBW2992/afatinib), mTOR (everolimus), and MEK (trametinib), we show that emerging drugs such as BEZ235 (panPI3K/mTOR), BKM-120 (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
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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 BKM120 have been reproduced in human tumor xenograft models in mice, with these combinations showing acceptable toxicity profiles (manuscript in preparation). An important goal for any monotherapy or drug combination is the identification of predictors of response so that clinicians can select the patient population most likely to benefit from treatment. The screen described here represents the discovery phase of such a project and additional work is required to identify predictors of response with a certainty that can drive patient selection. For instance, the kinetics
Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identification of Combinatorial Drugs that Synergistically Kill both EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.
Rickles, et al. (2015)
of the appearance of combination activity should be explored, and the relationships, if any, of synergy/non-synergy to intrinsic or acquired resistance should be further defined. To power such analyses, additional cell lines could be screened to generate a larger data set for more detailed genetic characterization. Additional analyses using larger data sets with sufficient breadth of known genetic profiles will be required to generate a fully vetted understanding of response prediction.
3. Smith JA, Wilson L, Azarenko O, Zhu X, Lewis BM, et al. (2010) Eribulin binds at microtubule ends to a single site on tubulin to suppress dynamic instability. Biochemistry 49:1331–1337.
In addition to identifying drug synergies, we have also identified antagonistic combinations. For example, low concentrations of cytarabine or topotecan have little effect on proliferation as single agents, but can dramatically antagonize eribulin activity under combination conditions. Indeed, this is not without precedent: antagonism has been observed for a variety of cancer drug combinations in preclinical studies, prompting follow up analyses to investigate both the mechanisms of the synergies as well as the effects of drug combination sequencing [28-35]. For instance, in one extensively studied example, the anthracycline doxorubicin antagonized activity of the vinca alkaloid vincristine in 83% (15/18) of hematopoietic cell lines tested. This antagonism was reproduced in vivo in a xenograft model and was also observed in 34% (12/35) of childhood leukemia cells simultaneously treated with both drugs ex vivo [35]. Moreover, combination activity was shown to be sequence dependent: if cells are exposed to the anthracycline first, antagonism is observed, but if exposure to vinca alkaloid precedes anthracycline, the combination result is beneficial. A p53-dependent mechanism for this antagonism was proposed, where anthracycline effects stabilize anti-apoptotic BCL2 family members, thus reducing cytotoxic effects of the vinca alkaloid. With respect to our current study, the eribulin antagonisms observed with 17-DMAG, cytarabine, topotecan and gemcitabine do not necessarily require that p53 be functional as was the case in the preceding example, but further study will be required to determine this. Indeed, the mechanisms by which these compounds antagonize eribulin may overlap or be entirely unrelated.
6. Swami U, Chaudhary I, Ghalib MH, Goel S. (2012) Eribulin--A review of preclinical and clinical studies. Crit Rev Oncol Hemat 81:163–184.
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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Citation: Rickles RJ, Matsui J, Zhu P, Funahashi Y, Grenier JM, et al. (2015) Identification of Combinatorial Drugs that Synergistically Kill both EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.
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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.
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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 EribulinSensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017.