Global Journal of Cancer Therapy eertechz

Richard J Rickles1, Junji Matsui3, Ping Research Article Zhu1, Yasuhiro Funahashi2,3, Jill M Grenier1, Janine Steiger1, Nanding Zhao2, Bruce A Littlefield2, Kenichi Identification of Combinatorial Nomoto2,3 and Toshimitsu Uenaka2* 1Horizon Discovery Inc., MA, Japan Drugs that Synergistically Kill both 2Eisai Inc., MA, Japan 3Eisai Co., Ltd., Japan Eribulin-Sensitive and Eribulin- Dates: Received: 17 October, 2015; Accepted: 03 November, 2015; Published: 04 November, 2015 Insensitive Tumor Cells *Corresponding author: Toshimitsu Uenaka, Ph.D., Executive Director, Production Creation Headquarter, Oncology & Antibody Drug Strategy, CINO (Chief Abstract Innovation Officer), E-mail: Eribulin sensitivity was examined in a panel of twenty-five human cancer cell lines representing a www.peertechz.com 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 standard of care. Chemotherapeutic drugs are often most effective when given in combination, in particular when synergistic killing is Eribulin mesylate, a microtubule dynamics inhibitor with a achieved without additive toxicity. To date, potential combination mechanism distinct from most other anti-tubulin therapeutics, is therapies for eribulin have been explored with only a limited number approved in the United States and many other countries for treatment of anti-cancer agents. We therefore have performed an in vitro of certain patients with advanced or metastatic breast cancer [1,2]. study of eribulin combined with 34 anticancer agents in 25 different Eribulin works by binding to exposed beta-tubulin subunits at the tumor cell lines of various types, through use of a combination high plus ends of microtubules, where it acts through end-poisoning throughput screening (cHTS) platform. Our goals were to identify mechanisms to inhibit microtubule growth and sequester tubulin drugs that might be paired with eribulin to increase clinical efficacy in into non-functional aggregates. In mitosis, this results in G2/M cell metastatic breast cancer, to identify drugs that convert eribulin non- cycle arrest, irreversible mitotic blockade, and ultimately cell death responders to responders, and to identify new therapeutic indications by apoptosis [3]. for eribulin utilization. Several compelling synergy effects with Eribulin has broad anticancer activity in a wide variety of approved and emerging drugs were observed. The preclinical data preclinical cancer models; as a result, numerous clinical trials of its provides insights about future clinical development strategies. effectiveness under mono therapy conditions are ongoing in other Materials and Methods non-breast tumor types [4-9]. Although eribulin failed to show meaningful activities in clinical trials of head and neck, pancreatic Materials and advanced non-small cell lung cancer [10,11], improvements in Cell lines were purchased from European Collection of Cell overall survival by eribulin were reported in a Phase 3 trial in advanced Cultures of Public Health England (A2780), Japanese Collection of soft tissue sarcoma compared with dacarbazine12 [ ], pointing to Research Bioresouces Cell Bank of the National Institute of Biomedical additional clinical uses for eribulin. Clinical trials are ongoing to Innovation (SNG-M), German Collection of Microorganisms and evaluate eribulin effectiveness for treatment of osteosarcoma, ovarian, Cell Cultures (KYSE-410), and American Type Culture Collection cervical, urothelium, brain metastasis, metastatic salivary gland and (all other cell lines). All cell lines are included in the Cancer Cell Line pediatric cancers with the promise that additional cancer indications Encyclopedia [13]. Cell culture media, fetal bovine serum, and cell will be identified. culture supplements were purchased from Gibco Life Technologies In certain ways, the clinical experience with eribulin has been (Thermo Scientific). Chemical matter was purchased from Enzo Life similar to that of other chemotherapies: mono therapy benefits Sciences, Inc., Micro Source Discovery Systems, Sigma-Aldrich Co. tend to be limited and it is often difficult to surpass the benefits of 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 Eribulin- Sensitive 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 Score equation integrates the experimentally-observed activity ATP Lite 1step Luminescence Assay System (Perkin Elmer). volume at each point in the matrix in excess of a model surface numerically derived from the activity of the component agents using Methods the Loewe model for additivity. Additional terms in the Synergy Cells were thawed and grown in culture media according to Score equation are used to normalize for various dilution factors used vendor’s recommendations. Detailed methods on the combination for individual agents and to allow for comparison of synergy scores assay were reported in [14]. Combinations were analyzed using across an entire experiment. Synergy Score and Loewe Volume Score, as described in [15,16] using Loewe Volume is an additional combination model score used to Horizon’s proprietary ChaliceTM Analyzer software. assess the overall magnitude of the combination interaction in excess Combination High-Throughput Screening of the Loewe additivity model. Loewe Volume is particularly useful when distinguishing synergistic increases in a phenotypic activity Cells were seeded in 384-well and 1536-well tissue culture treated (positive Loewe Volume) versus synergistic antagonisms (negative assay plates at cell densities ranging from 100 to 500 cells per well. Loewe Volume). Please see reference 13 for more detail. Twenty-four hours after cell seeding, compounds were added to assay plates with multiple replicates. Compounds were added to cells Self-cross based combination screening analysis using a 6x6 dose matrix, formed by six treatment points (including In order to objectively establish hit criteria for the combination DMSO control) of Eribulin and each combination partner as single screen analysis, twelve compounds were selected to be self-crossed agents and twenty-five combination points. Please see reference 12 across the twenty-five cell line panel as a means to empirically for additional detail. Concentration sampling ranges were selected determine a baseline additive, non-synergistic response. The identity after review of single agent activity of each molecule across the cell of the twelve self-cross compounds was determined by selecting line panel. Generally, concentrations between the EC10 and EC90 compounds with a variety of maximum response values and single were sampled. agent dose response steepness. Those drug combinations which At the time of treatment, a set of assay plates (which do not yielded effect levels that statistically superseded those baseline receive treatment) were collected and ATP levels were measured additivity values were considered synergistic. The Synergy Score by adding ATPLite. These Vehicle-zero (V0) plates were measured measure was used for the self-cross analysis. Synergy Scores of self- using an EnVision Multilabel Reader (Perkin Elmer). Treated assay crosses are expected to be additive by definition and, therefore, plates were incubated with compound for seventy-two hours. After maintain a synergy score of zero. However, while some self-cross seventy-two hours, treated assay plates were developed for endpoint Synergy Scores are near zero, many are greater suggesting that analysis using ATPLite. All data points were collected via automated experimental noise or non-optimal curve fitting of the single agent processes; quality controlled; and analyzed using Horizon’s dose responses are contributing to the slight perturbations in the proprietary software. score. Given the potential differences in cell line sensitivity to the eribulin combination activities, we chose to use a cell-line centric Horizon utilizes Growth Inhibition (GI) as a measure of cell strategy for the self-cross based combination screen analysis, focusing viability. The cell viability of vehicle is measured at the time of dosing on self-cross behavior in individual cell lines versus global review of (V ) and after seventy-two hours (V). A GI reading of 0% represents 0 the cell line panel activity. Combinations where the Synergy Score is no growth inhibition - cells treated with compound (T) and V vehicle greater than the mean self-cross plus two standard deviations (2σ’s) signals are matched. A GI 100% represents complete growth inhibition or three standard deviations (3 σ’s) can be considered candidate - cells treated by compound and V vehicle signals are matched. Cell 0 synergies at the 95% and 99% confidence levels, respectively. numbers have not increased during the treatment period in wells with GI 100% and may suggest a cytostatic effect for compounds reaching Results a plateau at this effect level. A GI 200% represents complete death of Evaluation of eribulin potency using a cell-based all cells in the culture well. Compounds reaching an activity plateau of assay GI 200% are considered cytotoxic. Horizon calculates GI by applying the following test and equation: Eribulin’s antiproliferative activity was assessed in a panel of twenty-five human cancer cells lines representing a variety of tumor TV− 0 If :

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. 010 Rickles, et al. (2015) in Figure 1 were included in the subsequent combination screen, The strategy of using target or pathway cluster profiles with visual with the following rationale: inclusion of eribulin sensitive cell lines inspection of matrices provides additional evidence linking a would provide models to identify drugs with the potential to increase particular target with combination activity, and helps to highlight eribulin efficacy, while inclusion of insensitive lines would facilitate subtle synergies that might otherwise be overlooked or insufficiently identification of drugs capable of converting eribulin insensitive cells revealed due to steep single agent dose response curves. A Synergy to eribulin sensitive cells. Score heat map for select target/pathway clusters is displayed in Figure 2, with cell lines clustered first according to eribulin sensitivity/ Receptor tyrosine kinase target-based cluster insensitivity then further demarcated based on tumor type. analysis As an example of enhancer library mechanistic redundancy, The thirty-five compound enhancer librarySupp ( lemental Table the combination screen contained three drugs known to inhibit 1) was screened in combination with eribulin in the twenty-five cell ErbB1/ErbB2 (EGFR/HER2) in cell-based assays: lapatinib, BIBW- lines described above in Figure 1. In order to objectively establish hit 2992 and erlotinib [18-21]. BIBW-2992 exhibited the best breadth criteria for the combination screen analysis, twelve compounds were of combination activity. The mechanism of action of BIBW-2992 selected to be self-crossed across the twenty-five cell line panel as a (irreversible inhibition) and higher potency for ErbB1 and ErbB2 means to empirically determine a baseline additive, non-synergistic may explain the greater breath-of-activity. Alternatively, synergies response. The identity of the twelve self-cross compounds was observed with BIBW-2992 but not lapatinib or erlotinib may be determined by selecting compounds with a variety of maximum the result of unique polypharmacy with BIBW-2992 inhibiting response values and single agent dose response steepness. Those drug secondary targets not affected by the other ErbB1/ErbB2 inhibitors. combinations which yielded effect levels that statistically superseded Representative Growth Inhibition and Loewe Excess dose matrices those baseline additivity values were considered synergistic. for some of the lapatinib combinations are shown in Figure 3 with A summary matrix view heat map of combination drug Synergy examples of erlotinib and BIBW-2992 combination activities shown Scores which exceed the cell line specific self-cross thresholds at in Supplemental Figures 3, 4. Low micromolar concentrations of 2σ’s above the mean is shown in Supplemental Figure 1. Using this lapatinib and erlotinib have been observed in pharmacokinetic strategy, 19.7% of the combinations exhibited some synergistic studies [18,19] suggesting the potential for reproducing the observed activity at the 95% confidence level. Using a more stringent criteria preclinical synergy here in a clinical setting. of 3σ cutoff (99% confidence), synergy was observed for 12.5% of the PI3K pathway inhibitors combinations (Supplemental Figure 2). Dysregulation of the PI3K pathway can transform cells by virtue The 35 compound enhancer library utilized contains some of constitutive activation and ultimately, stimulation of cellular redundancy in terms of targets and pathway inhibitors. Whenever proliferation and suppression of pro-apoptotic signaling. Four PI3K possible, target information was used to cluster similarly annotated pathway inhibitors were included in the screen. AZD8055 is a potent, enhancers across the cell line panel, and the Loewe Excess values selective, and orally bioavailable ATP-competitive mTOR kinase for Growth Inhibition matrices with high-to-moderate synergy inhibitor (TORC1 and TORC2); everolimus, an allosteric mTOR scores were individually reviewed to evaluate combination activities. (TORC1) inhibitor; BEZ235, a dual pan-PI3K/mTOR inhibitor; and

35.00 35.00 25.00 25.00 15.00 15.00 5.00 5.00 1.0 1.0 0.9 0.9

(nM ) 0.8 (nM ) 0.8 50 50 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 EribulinGI EribulinGI 0.2 0.2 0.1 0.1 0.0 0.0 T24 T24 A549 A375 T47D PC-3 786-0 FaDu A375 A549 MCF7 A2780 PC-3 T47D 786-0 FaDu A2780 MCF7 SNG-M Hep G2 SNG-M HT-1080 KATO III KATO KATOIII Hep G2 SK-BR-3 NCI-H69 SK-OV-3 HT-1080 SK-OV-3 SK-BR-3 NCI-H69 NCI-H460 NCI-H522 NCI-H526 KYSE-410 KYSE-410 NCI-H522 NCI-H526 NCI-H460 NCI-H1650 MIA PaCa-2 MIA NCI-H1650 MIA PaCa-2 MIA MDA-MB-231 MDA-MB-436 MDA-MB-468 MDA-MB-468 MDA-MB-436 MDA-MB-231 Lung Liver Breast Kidney Ovarian Bladder Prostate Stomach Pancreas Melanoma Head+Neck Esophageal Endometrial Fibrosarcoma

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. 011 Rickles, et al. (2015)

Eribulin-Sensitive Cell Lines Eribulin-Insensitve Cell Lines T24 PC-3 A375 A549 FaDu T47D 786-0 MCF7 A2780 SNG-M HepG2 KATOIII SK-BR-3 SK-OV-3 NCI-H69 HT-1080 KYSE-410 NCI-H460 NCI-H526 NCI-H522 NCI-H1650 MIA MIA PaCa-2 MDA-MB-231 Target Eribulin + MDA-MB-436 MDA-MB-468 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 ErbB1/ErbB2 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 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 PI3K Pathway 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 MEK ANDI(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.

MCF7 MDA-MB-468 NCI-H460 Dose MatrixNone | 500 | MCF7 | Loewe ExcessNone | 500 | MCF7 Dose MatrixNone | 100 | MDA- Loewe ExcessNone | 100 | MDA- Dose MatrixNone | 100 | NCI- Loewe ExcessNone | 100 | NCI-

ATP Lite | 72h | DT-1308668 | ATP Lite | 72h | DT-1308668 MB-468 | ATP Lite | 72h |DT-1308665 MB-468 | ATP Lite | 72h |DT-1308665 H460 | ATP Lite | 72h | DT-1308664 H460 | ATP Lite | 72h | DT-1308664 132 156 161 181 188 194 4 27 32 52 59 65 120 173 187 192 194 196 11 59 64 50 34 29 29 96 100 103 111 142 0 67 53 31 18 36

46 88 92 114 127 151 -3 -9 -5 18 31 54 56 91 139 172 179 181 -19 -1 25 30 19 14 5 94 100 119 111 120 0 65 53 48 18 14

24 73 71 82 102 118 22 -19 -26 -15 5 21 34 86 128 154 170 174 -2 16 23 12 10 7 -4 65 98 135 130 121 -4 37 51 64 37 15

19 75 77 77 100 114 19 -7 -20 -20 3 17 31 80 113 156 167 174 17 22 11 14 7 7 -4 49 84 107 121 118 -4 22 38 36 28 12

-4 68 75 82 89 120 -4 -1 -22 -14 -7 23 10 65 102 140 169 165 6 13 0 -2 9 -2 6 27 53 79 99 120 6 1 6 8 6 14 Lapatinib (uM) Lapatinib (uM) Lapatinib (uM) Lapatinib (uM) Lapatinib Lapatinib (uM) Lapatinib (uM) Lapatinib 0 .12 .37 1.1 3.3 9.9 0 .12 .37 1.1 3.3 9.9 0 .12 .37 1.1 3.3 9.9 0 .12 .37 1.1 3.3 9.9 - 69 76 81 93 115 0 .12- .37 1.16 3.3 9.9 -21 -16 -3 18 - 54 90 150 164 165 0 .12- .37 1.16 3.3 9.9 -10 8 3 -2 - 38 49 55 91 114 - 12 2 -17 -1 8 Growth Inhibition (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth Growth Inhibition Growth (%) Vol=2.46(.26) Chi2=66 Inhibition Growth (%) Vol=4.7(.48) Inhibition Growth (%) Vol=7.77(.5) Chi2=270 Chi2=330

0 .0012 .0037 .011 .033 .1 0 .0012 .0037 .011 .033 .1 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 0 .0012 .0037 .011 .033 .1 0 .0012 .0037 .011 .033 .1 Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) A2780 SNG-M Hep G2 Dose MatrixNone | 100 | A2780 Loewe ExcessNone | 100 | Dose MatrixNone | 100 | SNG-M Loewe ExcessNone | 100 | SNG- Dose MatrixNone | 100 | Hep G2 Loewe ExcessNone | 100 | Hep

| ATP Lite | 72h | DT-1308670 A2780 | ATP Lite | 72h | DT-1308670 | ATP Lite | 72h | DT-1308667 M | ATP Lite | 72h | DT-1308667 | ATP Lite | 72h | DT-1308669 G2 | ATP Lite | 72h | DT-1308669 86 154 174 176 180 191 8 65 38 40 43 54 62 74 90 137 152 151 2 12 24 61 44 24 75 115 117 128 135 131 0 39 41 53 59 54

30 98 127 122 136 159 -13 8 -9 -15 -1 22 36 57 86 135 145 146 -1 11 29 58 38 19 7 69 82 88 82 70 0 43 43 34 17 -2

25 93 133 130 138 158 16 -1 -4 -7 1 21 6 42 64 113 148 149 -6 13 16 36 41 22 11 48 78 85 82 76 11 33 49 39 21 5

3 92 121 110 139 160 2 -2 -15 -27 2 23 20 14 63 83 136 150 17 -9 18 7 29 22 -3 33 48 76 71 86 -3 20 23 31 11 16

13 93 115 132 136 156 12 6 -22 -4 -1 19 5 25 34 58 73 94 5 13 8 15 13 24 Lapatinib (uM) Lapatinib Lapatinib (uM) Lapatinib 5 32 66 75 126 147 4 13 23 -1 19 20 Lapatinib (uM) Lapatinib Lapatinib (uM) Lapatinib Lapatinib (uM) Lapatinib Lapatinib (uM) Lapatinib 0 .12 .37 1.1 3.3 9.9 0 .12 .37 1.1 3.3 9.9 0 .12 .37 1.1 3.3 9.9 0 .12 .37 1.1 3.3 9.9 0 .12 .37 1.1 3.3 9.9 - 90 108 123 144 153 - 5 -29 -14 8 16 0 .12 .37 1.1 3.3 9.9 - 19 21 42 56 73 - 7 -4 -1 -4 4

Growth Inhibition (%)N=2 Inhibition Growth - 7 50 80 94 131 - -11 9 5 -14 4 Growth Inhibition Growth (%) Vol=2.33(.51) Chi2=6.1 Growth Inhibition (%) N=3-4 Inhibition Growth Chi2=20 Growth Inhibition Growth (%) Vol=7.01(.44) Growth Inhibition (%)N=2 Inhibition Growth Growth Inhibition Growth (%) Vol=5.87(.48) Chi2=37

0 .0012 .0037 .011 .033 .1 0 .0012 .0037 .011 .033 .1 0 1.4e-4 4.1e-4 .0012 .0037 .011 0 1.4e-4 4.1e-4 .0012 .0037 .011 0 .0012 .0037 .011 .033 .1 0 .0012 .0037 .011 .033 .1 Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) KYSE-410 FaDu 786-0

Dose MatrixNone | 100 | KYSE- Loewe ExcessNone | 100 | KYSE Dose MatrixNone | 100 | FaDu | Loewe ExcessNone | 100 | FaDu Dose MatrixNone | 100 | 786-0 | Loewe ExcessNone | 100 | 786-

410 | ATP Lite | 72h | DT-1308666 -410 | ATP Lite | 72h | DT-1308666 ATP Lite | 72h | DT-1308660 | ATP Lite | 72h | DT-1308660 ATP Lite | 72h | DT-1308661 0 | ATP Lite | 72h | DT-1308661 181 186 193 192 193 185 24 29 36 34 36 27 109 149 183 185 184 187 12 53 86 88 88 91 91 167 173 189 191 197 0 74 78 91 89 92

74 73 91 107 145 160 -41 -42 -24 -8 30 45 90 109 170 165 167 174 -6 12 74 68 71 78 10 78 89 113 134 117 0 49 44 42 45 13

62 69 74 96 143 154 -1 -1 -9 7 53 64 90 98 149 168 164 175 -6 2 52 72 67 79 1 60 97 118 129 113 1 44 65 61 45 9

48 37 57 91 116 139 22 1 -1 7 26 49 82 93 135 160 142 174 -15 -3 38 63 46 78 0 36 78 102 113 129 0 23 51 48 31 27 24 28 56 79 105 115 15 10 11 -3 15 25 73 74 94 130 129 146 3 -15 -1 35 33 51 Lapatinib (uM) Lapatinib Lapatinib (uM) Lapatinib Lapatinib (uM) Lapatinib Lapatinib (uM) Lapatinib -2 25 44 76 94 126 -2 13 17 24 12 23 Lapatinib (uM) Lapatinib Lapatinib (uM) Lapatinib 0 .12 .37 1.1 3.3 9.9 0 .12 .37 1.1 3.3 9.9 0 .12 .37 1.1 3.3 9.9 0 .12 .37 1.1 3.3 9.9 0 .12 .37 1.1 3.3 9.9 - -3 37 80 78 97 - -7 2 -1 -12 6 - -1 53 85 88 102 - -6 2 -6 -8 6 0 .12 .37 1.1 3.3 9.9 Growth Inhibition (%)N=2 Inhibition Growth Growth Inhibition (%)N=2 Inhibition Growth Growth Inhibition Growth (%) Vol=4.13(.49) Chi2=99 Inhibition Growth (%) Vol=13.1(.41) Chi2=270 - 27 32 42 71 112 - 15 5 -10 -10 9 Growth Inhibition (%)N=2 Inhibition Growth Growth Inhibition Growth (%) Vol=11.1(.35) Chi2=190 4.6e-5 1.4e-4 4.1e-4 4.6e-5 1.4e-4 4.1e-4 1.4e-4 4.1e-4 1.4e-4 4.1e-4 0 .0012 .0037 0 .0012 .0037 0 .0012 .0037 .011 0 .0012 .0037 .011 0 .0012 .0037 .011 .033 .1 0 .0012 .0037 .011 .033 .1 Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) T24 A375 Dose MatrixNone | 100 | T24 | Loewe ExcessNone | 100 | T24 | Dose MatrixNone | 100 | A375 | Loewe ExcessNone | 100 | A375

ATP Lite | 72h | DT-1308662 ATP Lite | 72h | DT-1308662 ATP Lite | 72h | DT-1308663 | ATP Lite | 72h | DT-1308663 61 98 105 127 140 135 0 37 41 59 70 45 66 86 97 135 142 166 1 19 29 66 64 74

1 73 107 127 135 126 0 69 96 92 65 36 22 72 90 105 121 118 -1 33 38 40 43 26

9 44 83 101 124 126 9 44 81 84 57 36 6 63 87 110 122 123 5 43 50 51 44 31

-4 1 40 79 94 109 -4 1 39 65 30 19 7 41 77 96 106 115 7 25 43 39 29 23

-7 -6 -1 54 76 111 -7 -6 -2 42 11 21 -3 38 52 75 96 119 -3 23 20 19 18 27 Lapatinib (uM) Lapatinib (uM) Lapatinib (uM) Lapatinib Lapatinib (uM) Lapatinib 0 .12 .37 1.1 3.3 9.9 0 .12 .37 1.1 3.3 9.9 0 .12 .37 1.1 3.3 9.9 - -6 -4 13 63 91 - -7 -5 2 -1 1 - 20 30 53 74 96 0 .12- .37 1.16 3.3 9.9 -1 -2 -4 5 Growth Inhibition (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth Chi2=150 Chi2=80 Growth Inhibition Growth (%) Vol=11.3(.55) Inhibition Growth (%) Vol=9.22(.44)

0 .0012 .0037 .011 .033 .1 0 .0012 .0037 .011 .033 .1 0 1.4e-4 4.1e-4 .0012 .0037 .011 0 1.4e-4 4.1e-4 .0012 .0037 .011 Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) 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 (Figure 4). Combination activity is selective (for example, little or no more of these inhibitors are synergistic in combination with eribulin combination activity in SK-BR-3, NCI-H552, NCI-H526, Mia PaCa- in the majority of cell lines in the panel. For a subset of cell lines, 2, and 786-0) pointing to specific genetic determinants in responsive synergy is observed with all four PI3K pathway inhibitors. The cell lines as being important for combination activity. breadth of activity observed in the cell line panel and for the various MEK inhibitors PI3K pathway inhibitors highlight the importance of PI3K signaling for tumor cell proliferation and/or survival upon eribulin exposure 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 Eribulin- Sensitive and Eribulin-Insensitive Tumor Cells. Glob J Cancer Ther 1(1): 009-017. 012 Rickles, et al. (2015)

Eribulin-Sensitive Cell Lines Eribulin-Insensitve Cell Lines T24 PC-3 A549 A375 FaDu T47D 786-0 MCF7 A2780 SNG-M KATOIII G2 Hep SK-BR-3 NCI-H69 SK-OV-3 HT-1080 KYSE-410 NCI-H460 NCI-H522 NCI-H526 NCI-H1650 MIA PaCa-2 MIA MDA-MB-231 Target Eribulin + MDA-MB-436 MDA-MB-468 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 PI3K Pathway 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 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 Everolimus

MDA-MB-468 NCI-H69 A2780 FaDu HT-1080 T47D Dose Matrix | None | 100 | Dose Matrix | None | 100 | MDA Dose Matrix | None | 500 | NCI- Dose Matrix | None | 100 | Dose Matrix | None | 100 | FaDu Dose Matrix | None | 100 | HT- T47D | ATP Lite | 72h DT-1308695 -MB-468 | ATP Lite | 72h DT-1308694 H69 | ATP Lite | 72h DT-1308699 A2780 | ATP Lite | 72h DT-1308693 | ATP Lite | 72h DT-1308703 1080 | ATP Lite | 72h DT-1308706 100 128 126 141 141 127 47 61 121 174 184 179 132 132 149 159 164 169 103 152 166 170 177 178 73 73 106 146 144 178 87 117 142 155 179 184 99 109 130 132 129 125 26 62 100 157 180 174 104 104 143 163 162 166 97 137 158 167 177 184 56 66 94 106 145 162 77 91 110 147 176 185 .026 .078 81 104 116 113 128 111 17 55 71 132 159 162 57 84 123 159 165 168 90 126 150 157 173 182 25 39 74 103 130 166 57 70 92 140 174 187 56 80 103 112 106 92 AZD 8055 14 26 60 109 152 142 39 62 100 153 163 164 76 110 134 152 170 179 10 15 58 94 97 129 32 52 83 109 171 184 .026 .078 .23 .7 .026 .078 .23 .7 .026 .078 .23 .7 .026 .078 .23 .7 .026 .078 .23 .7 .0029 .0086 35 76 96 109 106 79 .0086

3 38 69 107 135 154 19 46 87 147 161 162 54 110 138 143 152 177 1 25 56 80 94 134 11 38 74 102 146 179 AZD8055 (uM) 9.6e-4 .0086 .0086 .0086 .0086 AZD8055 (uM) AZD8055 (uM) AZD8055 (uM) AZD8055 (uM) AZD8055 (uM)

0 - 71 100 100 94 66 0 0 0 0 0 - 52 64 106 136 150 - 21 61 130 140 150 - 91 104 108 128 147 - 3 54 89 95 102 - 14 64 86 135 174 (%)N=2 Inhibition Growth Growth Inhibition (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth Growth Inhibition (%) N=2-3 Inhibition Growth (%) N=1-2 Inhibition Growth 0 .0012 .0037 .011 .033 .1 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 0 1.4e-4 4.1e-4 .0012 .0037 .011 0 .0012 .0037 .011 .033 .1 0 1.4e-4 4.1e-4 .0012 .0037 .011 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Dose Matrix | None | 100 | Dose Matrix | None | 100 | MDA Dose Matrix | None | 500 | NCI- Dose Matrix | None | 100 | Dose Matrix | None | 100 | FaDu Dose Matrix | None | 100 | HT- T47D | ATP Lite | 72h DT-1308725 -MB-468 | ATP Lite | 72h DT-1308724 H69 | ATP Lite | 72h DT-1308726 A2780 | ATP Lite | 72h DT-1308727 | ATP Lite | 72h DT-1308723 1080 | ATP Lite | 72h DT-1308728 100 125 139 138 145 147 103 153 173 185 186 186 139 148 165 163 170 180 103 155 164 167 176 175 100 123 159 170 178 170 95 128 130 167 175 188 104 123 141 146 150 141 93 127 173 184 191 187 103 113 148 164 169 174 99 138 159 163 173 180 81 102 125 152 170 176 82 115 126 163 179 188 107 120 134 150 156 145 48 103 149 173 182 182 57 52 112 155 166 165 89 109 143 147 166 180 67 72 98 140 160 161 77 99 104 153 175 184 .012 .037 .11 .012 .037 .11 .012 .037 .11 .012 .037 .11 97 103 125 140 142 132 22 66 105 146 173 176 13 32 85 147 158 163 58 105 131 134 156 168 49 73 105 102 142 172 30 62 85 112 173 187 BEZ235

BEZ235(uM) 55 83 100 115 118 95 BEZ235(uM) BEZ235(uM) BEZ235(uM) BEZ235(uM) BEZ235(uM) 25 33 88 134 168 165 16 33 83 140 147 159 33 94 134 127 139 165 24 33 66 105 97 153 18 40 72 105 157 181 .0014 .0041 .0014 .0041 .0014 .0041 .0014 .0041

0 .012- .037 .11 .3360 1 99 102 90 67 0 0 0 0 .012 .037 .11 .33 1 0 - 61 82 120 143 158 - 15 58 126 144 146 - 89 108 115 133 159 - -4 52 84 91 104 - 22 57 96 151 180 (%) N=1-2 Inhibition Growth Growth Inhibition (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth Growth Inhibition (%) N=1-2 Inhibition Growth (%) N=1-2 Inhibition Growth 0 .0012 .0037 .011 .033 .1 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 0 1.4e-4 4.1e-4 .0012 .0037 .011 0 .0012 .0037 .011 .033 .1 0 1.4e-4 4.1e-4 .0012 .0037 .011 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM)

Dose Matrix | None | 100 | Dose Matrix | None | 100 | MDA Dose Matrix | None | 500 | NCI- Dose Matrix | None | 100 | Dose Matrix | None | 100 | FaDu Dose Matrix | None | 100 | HT- T47D | ATP Lite | 72h DT-1308718 -MB-468 | ATP Lite | 72h DT-1308717 H69 | ATP Lite | 72h DT-1308719 A2780 | ATP Lite | 72h DT-1308720 | ATP Lite | 72h DT-1308721 1080 | ATP Lite | 72h DT-1308722 94 129 127 141 128 132 104 121 156 165 168 164 164 170 171 171 175 174 122 161 169 181 177 185 84 106 142 141 158 164 85 99 145 161 171 186 32 92 106 127 120 104 6 54 99 142 150 163 74 93 140 160 167 168 75 125 148 147 160 179 23 66 84 95 113 162 19 59 77 119 174 180 19 79 99 123 104 83 13 16 72 148 146 155 34 58 98 148 158 160 30 109 130 132 151 166 9 43 75 86 100 128 8 35 70 96 141 178 13 57 108 113 107 67 BKM-120 -22 29 59 97 143 132 16 41 76 133 151 156 21 94 114 123 138 159 18 37 62 84 99 114 12 30 66 92 150 181 13 63 98 106 103 74

7 15 63 110 137 142 1 20 62 136 135 153 2 91 106 110 130 149 -3 44 60 81 91 111 -5 26 63 92 145 175 BKM-120(uM) BKM-120(uM) BKM-120(uM) BKM-120(uM) BKM-120(uM) BKM-120(uM)

0 .025 .074- .22 .6663 2 90 101 98 72 0 .025 .074 .22 .66 2 0 .025 .074 .22 .66 2 0 .025 .074 .22 .66 2 0 .025 .074 .22 .66 2 0 .025 .074 .22 .66 2 - 45 64 106 136 142 - 27 62 126 143 147 - 90 100 109 129 143 - 6 54 89 92 99 - 25 56 89 124 163 (%)N=2 Inhibition Growth Growth Inhibition (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth Growth Inhibition (%) N=1-2 Inhibition Growth Growth Inhibition (%) N=2-3 Inhibition Growth 0 .0012 .0037 .011 .033 .1 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 0 1.4e-4 4.1e-4 .0012 .0037 .011 0 .0012 .0037 .011 .033 .1 0 1.4e-4 4.1e-4 .0012 .0037 .011 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM)

Dose MatrixNone | 100 | T47D | Dose MatrixNone | 100 | MDA- Dose MatrixNone | 500 | NCI- Dose MatrixNone | 100 | A2780 Dose MatrixNone | 100 | FaDu | Dose MatrixNone | 100 | HT- ATP Lite | 72h | DT-1308897 MB-468 | ATP Lite | 72h |DT-1308898 H69 | ATP Lite | 72h | DT-1308896 | ATP Lite | 72h | DT-1308899 ATP Lite | 72h | DT-1308895 1080 | ATP Lite | 72h | DT-1308900 32 84 106 129 106 102 64 119 158 185 187 187 35 56 106 150 162 166 74 111 147 151 169 179 44 58 73 101 132 136 51 76 107 147 175 175 31 74 105 111 118 96 39 117 162 176 185 183 23 53 86 151 163 164 62 110 148 141 163 180 42 59 75 95 108 147 48 82 112 144 177 185 .017 .05 .017 .05 .017 .05 15 74 106 120 118 81 25 84 130 163 185 186 20 33 76 139 146 160 30 97 136 136 159 173 34 43 72 89 98 142 54 73 97 144 169 180 36 75 108 117 114 90 2 72 127 157 172 177 14 26 72 134 151 155 14 90 123 123 143 161 37 57 68 89 103 128 50 70 104 145 173 184 .0019 .0055 .0019 .0055 .0019 .0055 Everolimus 29 83 112 111 104 90 7 81 91 154 171 168 14 21 65 134 150 145 4 90 111 130 147 159 33 35 77 92 93 128 53 74 96 153 171 182 6.2e-4 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 7.6e-6 2.3e-5 6.8e-5 2.1e-4 6.2e-4 6.2e-4 Everolimus (uM) Everolimus 6.2e-4 Everolimus (uM) Everolimus (uM) Everolimus (uM) Everolimus (uM) Everolimus Everolimus (uM) Everolimus 0 - 51 88 104 94 73 0 0 0 0 0 - 54 90 150 164 165 - 12 66 121 133 135 - 90 108 123 144 153 - -1 53 85 88 102 - 43 54 122 150 168 (%) N=1-2 Inhibition Growth Growth Inhibition (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth Growth Inhibition (%)N=2 Inhibition Growth 0 .0012 .0037 .011 .033 .1 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 0 1.4e-4 4.1e-4 .0012 .0037 .011 0 .0012 .0037 .011 .033 .1 0 1.4e-4 4.1e-4 .0012 .0037 .011 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) 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 be used to gauge both synergies and antagonisms. A Loewe Volume and Loewe Excess dose matrices are shown in Figure 5. There is good heat map is shown in Figure 7 for 17-DMAG, cytarabine, topotecan, concordance for both inhibitors; when synergy is observed with and gemcitabine, with greater negative values (antagonism) one of the MEK inhibitors, it is most often observed with the other. highlighted in increasingly darker shades of blue and increasing MEK combination activity was strongest in MDA-MB-231, A2780 positive values (synergies) highlighted in increasingly darker shades and Hep G2. For some cell lines, synergy is observed when eribulin of red. Also shown are representative combination dose matrices is combined with either PI3K pathway inhibitors or MEK inhibitors for selected antagonistic activities observed. The 17-DMAG Loewe (for example, FaDu, NCI-H460 and A2780). For other cell lines, Volume combination activity pattern is complex: for 6 cell lines (red (MCF7, NCI-H69, KYSE-410, HT-1080 and T47D), synergies are shaded), combinations with eribulin are synergistic, while for 4 cell PI3K pathway-specific, with no synergies seen for MEK inhibitors. lines, antagonism was observed. BCL-2 Inhibition Discussion ABT-263 (Navitoclax) is a potent Bcl-xL, Bcl-2, and Bcl-w inhibitor currently in multiple clinical trials as a mono therapy or Eribulin has broad anticancer activity in a wide variety of in combination with approved drugs. Strong synergies with eribulin preclinical cancer models [4,7] and numerous monotherapy and good breadth of activity was observed with ABT-263, in both clinical trials are ongoing to investigate efficacy in non-breast eribulin-sensitive and insensitive cell lines (Figure 6). Strong synergy tumor types (see www.clinicaltrials.gov). Consistent with utility was observed in most but not all of the cell lines, suggesting that an for additional potential indications, a variety of tumor cell lines eribulin x ABT-263 combination is not non-selectively synergistically including lung, ovarian, endometrial, head and neck, prostate and toxic. melanoma are shown here to be sensitive to eribulin at clinically relevant concentrations. Since tumor cells have a robust capacity to Antagonistic drug pairings lessen the therapeutic effects of cancer drugs through adaptive and As described in Materials and Methods, Loewe Volume values can acquired resistance mechanisms, the identification of drugs that can

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. 013 Rickles, et al. (2015)

NCI-H460 A2780 Hep G2 Dose Matrix | None | 100 | NCI- Loewe Excess | None | 100 | Dose Matrix | None | 100 | Loewe Excess | None | 100 | Dose Matrix | None | 100 | Hep Loewe Excess | None | 100 |

H460 | ATP Lite | 72h DT-1308817 NCI-H460 | ATP Lite | 72hDT-1308817 A2780 | ATP Lite | 72h DT-1308813 A2780 | ATP Lite | 72h DT-1308813 G2 | ATP Lite | 72h DT-1308818 Hep G2 | ATP Lite | 72h DT-1308818 71 82 105 108 128 144 2 14 36 34 42 50 171 190 192 191 195 194 11 30 32 31 35 34 164 183 183 184 184 190 2 21 21 22 21 27

63 74 90 111 128 132 -4 7 21 37 42 38 138 187 187 189 191 192 -3 46 46 48 50 50 143 177 181 183 184 188 -3 30 34 37 37 42

55 76 87 89 98 117 0 16 21 15 12 22 94 162 165 174 177 184 -13 35 38 47 50 56 121 165 164 179 185 186 -3 41 41 56 61 62

19 55 58 79 97 121 1 9 -2 5 10 27 70 139 150 155 166 177 5 16 22 27 39 50 99 122 128 125 168 168 4 27 33 30 73 73 ANDI (E6201)

-1 33 44 64 86 101 -4 -6 -13 -10 -1 7 37 119 136 139 165 169 6 10 9 12 38 41 65 75 81 94 116 130 -1 8 14 28 48 61 ANDI(E6201)(uM) ANDI(E6201)(uM) ANDI(E6201)(uM) ANDI(E6201)(uM) ANDI(E6201)(uM) ANDI(E6201)(uM) 0 .12 .37 1.1 3.3 10 - 46 47 69 86 99 0 .12- .3711 1.1 3.3-8 10 -5 -1 5 0 .12- .3791 1.1 3.3104 10 108 128 147 0 .12- .37 1.16 3.3-24 10 -19 0 19 0 .12- .3716 1.1 3.314 10 46 47 66 0 .12- .37 1.16 3.3-8 10 7 -7 2 Growth Inhibition (%) N=1-2 Inhibition Growth Growth Inhibition (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth Growth Inhibition Growth (%) Vol=4.31(.43) Chi2=15 Inhibition Growth (%) Vol=8.91(.19) Chi2=580 Inhibition Growth (%) Vol=9.53(.86) Chi2=40

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

Dose MatrixNone | 100 | NCI- Loewe ExcessNone | 100 | NCI- Dose MatrixNone | 100 | A2780 Loewe ExcessNone | 100 | Dose MatrixNone | 100 | Hep G2 Loewe ExcessNone | 100 | Hep

H460 | ATP Lite | 72h | DT-1308808 H460 | ATP Lite | 72h | DT-1308808 | ATP Lite | 72h | DT-1308809 A2780 | ATP Lite | 72h | DT-1308809 | ATP Lite | 72h | DT-1308810 G2 | ATP Lite | 72h | DT-1308810 66 91 121 131 151 151 3 27 57 57 64 57 95 139 156 157 167 176 0 44 59 41 34 33 157 167 174 174 178 188 5 15 22 22 26 36

61 81 94 101 122 139 -2 18 30 27 36 45 89 127 151 156 161 173 -2 35 55 39 29 30 139 147 160 158 173 181 6 13 27 24 40 47 .0027 .0082 .0027 .0082

58 75 85 93 111 128 -5 12 22 20 24 34 84 112 141 145 158 171 1 24 45 29 26 28 89 110 114 142 134 158 -12 9 12 41 32 56 .025 .074 .22 .025 .074 .22 9.1e-4 9.1e-4 Trametinib 65 71 84 94 95 112 2 8 21 20 8 18 67 108 135 140 157 172 -1 27 41 24 24 29 56 65 77 87 98 131 -6 4 13 20 24 50

48 69 77 85 93 116 0 7 14 11 7 22 48 109 131 133 147 162 0 35 37 17 14 19 46 57 57 65 67 98 15 23 19 16 4 22 .0027 .0082 .0027 .0082 Trametinib (uM) Trametinib (uM) Trametinib (uM) Trametinib (uM) Trametinib (uM) Trametinib Trametinib (uM) Trametinib 0 1e-4 3e-4 0 .025 .074 .22 .66 2 0 0 0 1e-4 3e-4 - 46 47 69 86 99 0 .025- .074 .2211 .66 2 -8 -5 -1 5 - 89 100 101 118 149 - 20 6 -15 -15 6 - 9 16 37 50 77 - 3 0 3 -7 4 Growth Inhibition (%) N=1-2 Inhibition Growth Growth Inhibition (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth Chi2=53 Growth Inhibition Growth (%) Vol=8.17(.48) Inhibition Growth (%) Vol=6.16(.49) Chi2=67 Chi2=17 Growth Inhibition Growth (%) Vol=6.67(.45)

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

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

Dose Matrix | None | 100 | MDA Loewe Excess | None | 100 | Dose Matrix | None | 100 | NCI- Loewe Excess | None | 100 | Dose Matrix | None | 100 | KYSE Loewe Excess | None | 100 | -MB-231 | ATP Lite | 72h MDA-MB-231 | ATP Lite | 72h H1650 | ATP Lite | 72h NCI-H1650 | ATP Lite | 72h -410 | ATP Lite | 72h KYSE-410 | ATP Lite | 72h 180 180 183 187 191 183 14 12 16 20 24 16 140 165 175 196 198 197 13 39 48 69 71 71 178 194 195 197 195 199 25 40 41 43 41 45

100 109 135 177 180 187 -23 -13 13 55 58 64 60 53 80 167 193 193 -25 -36 -13 73 98 98 95 192 193 192 196 196 -32 65 67 65 69 69

70 85 90 152 177 181 11 20 14 70 92 95 47 52 85 146 194 198 9 -3 15 58 100 103 74 190 192 193 191 194 -18 98 100 101 99 102

15 66 79 146 179 174 -5 34 27 74 96 89 15 46 67 151 194 195 3 17 13 69 102 101 62 184 190 189 196 194 5 113 116 113 120 118 22 50 75 125 171 171 17 32 35 58 90 86 53 168 187 190 192 190 22 108 115 115 116 115 ABT-263(uM) ABT-263(uM) ABT-263(uM) ABT-263(uM) 29 22 74 117 190 194 26 4 28 36 98 99 ABT-263(uM) ABT-263(uM) 0 .62 1.2 2.5 5 10 0 .62 1.2 2.5 5 10 0 .62 1.2 2.5 5 10 0 .62 1.2 2.5 5 10 - 42 68 74 75 76 - 0 0 0 0 0 - 12 24 61 82 84 - 5 -3 1 3 -1 0 .62 1.2 2.5 5 10 0 .62 1.2 2.5 5 10 Growth Inhibition (%)N=2 Inhibition Growth Growth Inhibition (%) Vol=22(.36) Chi2=180 Inhibition Growth (%) Vol=22(.36) Growth Inhibition (%)N=2 Inhibition Growth Growth Inhibition Growth (%) Vol=11.8(.72) Chi2=110 - 1 41 73 91 95 - -7 5 -4 -1 1 Growth Inhibition (%)N=2 Inhibition Growth Growth Inhibition Growth (%) Vol=13.6(.49) Chi2=1200 1.4e-4 4.1e-4 .0012 .0037 1.4e-4 4.1e-4 .0012 .0037 0 .0012 .0037 .011 .033 .1 0 .0012 .0037 .011 .033 .1 0 .011 0 .011 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM)

MCF7 SK-BR-3 SNG-M

Dose Matrix | None | 500 | Loewe Excess | None | 500 | Dose Matrix | None | 500 | SK- Loewe Excess | None | 500 | SK Dose Matrix | None | 100 | SNG- Loewe Excess | None | 100 | MCF7 | ATP Lite | 72h MCF7 | ATP Lite | 72h BR-3 | ATP Lite | 72h -BR-3 | ATP Lite | 72h M | ATP Lite | 72h SNG-M | ATP Lite | 72h 102 124 110 125 131 132 7 29 15 29 35 37 76 103 122 139 134 135 6 27 37 7 -9 -9 41 63 78 86 160 166 0 21 36 18 69 66

69 113 122 125 128 128 -11 19 28 31 34 34 42 58 115 138 147 150 -7 -9 31 6 4 6 4 27 72 81 129 150 0 -8 31 13 38 50

57 109 112 114 122 133 -1 17 18 20 28 39 21 55 123 141 147 151 -1 3 39 9 4 6 -6 24 54 82 103 153 -6 1 13 14 12 52

37 94 107 110 122 129 0 5 13 16 28 35 12 31 119 138 147 148 6 -5 35 6 4 4 12 13 42 76 104 125 12 -5 1 8 13 24

25 83 91 99 110 121 5 -1 -3 5 16 27 13 36 100 140 146 149 12 6 15 8 3 5 19 11 64 78 94 125 19 -4 25 10 3 24 ABT-263(uM) ABT-263(uM) ABT-263(uM) ABT-263(uM) ABT-263(uM) ABT-263(uM) 0 .62 1.2 2.5 5 10 0 .62 1.2 2.5 5 10 0 .62 1.2 2.5 5 10 0 .019 .039 .078 .16 .31 0 .019 .039 .078 .16 .31 - 68 73 83 94 108 - 5 -21 -11 0 14 0 .62- 1.2 2.517 585 10 128 138 148 - -3 3 -4 -5 4 - 9 42 65 91 101 - -5 5 -3 0 0 Growth Inhibition (%)N=2 Inhibition Growth (%) N=1-2 Inhibition Growth (%)N=2 Inhibition Growth Growth Inhibition (%) Vol=5.56(.3) Chi2=29 Inhibition Growth (%) Vol=5.56(.3) Inhibition Growth (%) Vol=2.33(.36) Chi2=13 Chi2=8 Inhibition Growth (%) Vol=5.24(.53)

0 .0012 .0037 .011 .033 .1 0 .0012 .0037 .011 .033 .1 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 0 1.4e-4 4.1e-4 .0012 .0037 .011 0 1.4e-4 4.1e-4 .0012 .0037 .011 Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) 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). paired with eribulin to trigger selective and synergistic tumor cell melanoma therapy. However, the treatment of vemurafenib caused death represents a critical path toward optimizing patient benefit. resistance in some patients through its 2-18 months post treatment Specifically, drug combinations need to be identified for situations [22]. Recently, four articles reported five resistance mechanisms in which the effects of eribulin monotherapy are suboptimal, or in for vemurafenib [23-27]. Those authors attributed their resistance which intrinsic or acquired drug resistance might be overcome with mechanisms to the overexpression of PDGFR-β tyrosine kinase [23], strategically selected drug combinations. and/or Q61K NRAS [24], COT kinase [25], IGF-1R [26], C121S MEK [27], in melanoma cells. Selective inhibitors against those Not only eribulin, but also all anticancer drugs need to be looked molecules may be appropriate combination partners to prohibit these for appropriate combination partners. A molecular targeted agent, resistant mechanisms in V600E melanoma patients. Comprehensive vemurafenib (BRAF inhibitor), demonstrated 48% anti-tumor combination analysis by using comprehensive compounds on several response and extended PFS to 5.3 months in V600E melanoma cancer cells harboring different molecular mutation are very useful to patients in the BRIM3 study [21], in a first-line BRAF mutated find out appropriate combination partners.

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. 014 Rickles, et al. (2015)

Eribulin-Sensitive Cell Lines Eribulin-Insensitve Cell Lines T24 PC-3 A375 A549 T47D FaDu 786-0 MCF7 A2780 SNG-M HepG2 KATOIII SK-BR-3 NCI-H69 SK-OV-3 HT-1080 KYSE-410 NCI-H460 NCI-H522 NCI-H526 NCI-H1650 MIA MIA PaCa-2 MDA-MB-231 MDA-MB-436 Eribulin + MDA-MB-468 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

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

Dose Matrix | None | 100 | MDA Dose Matrix | None | 100 | MDA Dose Matrix | None | 500 | SK- Dose Matrix | None | 100 | KYSE Dose Matrix | None | 100 | HT- Dose Matrix | None | 500 |

-MB-436 | ATP Lite | 72h DT-1308936 -MB-468 | ATP Lite | 72h DT-1308941 BR-3 | ATP Lite | 72h DT-1308938 -410 | ATP Lite | 72h DT-1308939 1080 | ATP Lite | 72h DT-1308937 KATOIII | ATP Lite | 72hDT-1308940 69 59 69 72 68 64 69 86 77 86 89 91 98 101 94 105 119 125 51 55 44 56 56 54 101 111 111 116 128 136 13 18 10 13 12 5

36 60 41 64 63 63 47 62 58 78 64 78 96 89 102 98 106 113 39 46 52 53 55 44 78 81 90 91 95 111 -16 1 8 -4 -10 -3

13 24 28 42 63 63 34 53 50 60 52 62 90 91 98 99 111 111 40 44 44 44 50 50 52 49 63 76 87 116 -29 -28 -8 -22 -11 3

-7 11 25 62 89 108 23 17 44 73 82 70 74 80 90 98 107 109 44 35 43 49 42 59 14 20 42 75 110 156 -44 4 20 7 20 25 Cytarabine

-9 9 29 56 113 104 5 32 47 86 108 122 35 50 95 119 129 131 30 47 15 31 54 65 -8 5 35 82 139 180 -32 6 29 36 46 53 Cytarabine (uM) Cytarabine (uM) Cytarabine (uM) Cytarabine (uM) Cytarabine (uM) Cytarabine (uM) Cytarabine 0 .014 .041 .12 .37 1.1 0 .12 .37 1.1 3.3 9.9 0 .12 .37 1.1 3.3 9.9 0 .014 .041 .12 .37 1.1 - 7 43 74 115 120 - 53 64 106 136 150 0 .014 .041- .12 .3740 1.1 92 144 152 155 - -5 43 79 87 99 - 22 57 96 151 180 0 .12- .37 1.135 3.3 9.9 47 52 56 63 Growth Inhibition (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth Growth Inhibition (%) N=2-3 Inhibition Growth (%) N=1-2 Inhibition Growth

0 1.4e-4 4.1e-4 .0012 .0037 .011 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 0 1.4e-4 4.1e-4 .0012 .0037 .011 Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM)

MDA-MB-468 NCI-H1650 NCI-H69 A2780 KYSE-410 HT-1080 Dose MatrixNone | 100 | MDA- Dose MatrixNone | 100 | NCI- Dose MatrixNone | 500 | NCI- Dose MatrixNone | 100 | A2780 Dose MatrixNone | 100 | KYSE- Dose MatrixNone | 100 | HT-

H69 | ATP Lite | 72h | DT-1308947 MB-468 | ATP Lite | 72h |DT-1308946 H1650 | ATP Lite | 72h | DT-1308943 | ATP Lite | 72h | DT-1308942 410 | ATP Lite | 72h | DT-1308945 1080 | ATP Lite | 72h | DT-1308944 176 177 181 183 183 179 162 154 157 153 154 158 146 143 141 149 148 154 96 96 102 97 104 118 118 111 109 111 108 120 190 186 191 190 194 193

160 160 156 164 167 172 109 103 124 115 111 117 123 119 122 136 127 136 92 97 97 105 102 107 76 73 72 70 75 78 144 152 158 166 156 168

113 130 109 113 113 105 54 54 67 76 71 59 92 90 102 93 106 118 74 93 101 112 103 113 48 39 46 44 55 50 115 117 118 125 129 145 .014 .041 .12 Topotecan (uM) (uM) (uM) 53 63 71 69 73 82 28 30 27 34 41 63 73 67 66 82 91 98 (uM) 26 91 111 121 126 145 (uM) 25 26 28 40 37 53 (uM) 88 88 91 92 95 108

31 65 68 93 111 139 23 16 37 51 82 80 53 56 51 90 100 100 7 89 110 117 125 152 5 -5 27 64 89 92 59 71 74 78 84 90 .0015 .0046 0 .014 .041 .12 .37 1.1 Topotecan Topotecan Hydrochloride Topotecan Topotecan Hydrochloride 0 .014 .041 .12 .37 1.1 Topotecan Topotecan Hydrochloride 0 .014 .041- .12 .3753 1.1 64 106 136 150 - 12 24 61 82 84 - 20 63 121 130 130 Topotecan Hydrochloride 0 - 89 108 115 133 159 Topotecan Hydrochloride 0 .014 .041- .12 .37-5 1.1 43 79 87 99 Topotecan Hydrochloride 0 .12- .37 1.122 3.3 9.9 57 96 151 180 Growth Inhibition (%)N=2 Inhibition Growth Growth Inhibition (%) N=1-2 Inhibition Growth (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth (%)N=2 Inhibition Growth Growth Inhibition (%) N=2-3 Inhibition Growth

0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 0 1.4e-4 4.1e-4 .0012 .0037 .011 0 1.4e-4 4.1e-4 .0012 .0037 .011 0 .0012 .0037 .011 .033 .1 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 0 4.6e-5 1.4e-4 4.1e-4 .0012 .0037 Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) Eribulin(E7389) (uM) 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.

In this study, we describe the identification of approved and eribulin combination activities with erlotinib, everolimus, and BKM- emerging drugs that synergize when combined with eribulin, with 120 have been reproduced in human tumor xenograft models in good breadth of combination activity observed in the twenty five cell mice, with these combinations showing acceptable toxicity profiles line panel used for screening. Synergy is observed in both eribulin- (manuscript in preparation). sensitive and insensitive cell lines, suggesting potential clinical benefit An important goal for any monotherapy or drug combination is for both responders and non-responders of eribulin monotherapy. the identification of predictors of response so that clinicians can select In addition to approved drugs that target EGFR/HER2 (erlotinib, the patient population most likely to benefit from treatment. The lapatinib, BIBW2992/afatinib), mTOR (everolimus), and MEK screen described here represents the discovery phase of such a project (trametinib), we show that emerging drugs such as BEZ235 (pan- and additional work is required to identify predictors of response with PI3K/mTOR), BKM-120 (PI3Kα), and ABT-263 (BCL-2 family inhibitor) strongly synergize with eribulin in certain cell lines. a certainty that can drive patient selection. For instance, the kinetics Furthermore, the use of both target-specific (EGFR/HER2, MEK) of the appearance of combination activity should be explored, and the and pathway-specific (PI3K), mechanistically redundant compounds relationships, if any, of synergy/non-synergy to intrinsic or acquired in our studies validates the identified target specificities as being resistance should be further defined. To power such analyses, important for synergy. By evaluating a wide concentration range additional cell lines could be screened to generate a larger data set for these molecules, we can interrogate combination activities and for more detailed genetic characterization. Additional analyses using evaluate target selectivity; in addition, where pharmacokinetic data larger data sets with sufficient breadth of known genetic profiles will are available, we can obtain initial insights as to whether such effects be required to generate a fully vetted understanding of response could occur at clinically relevant concentrations. Looking at the prediction. eribulin’s concentrations which showed synergy effects combined In addition to identifying drug synergies, we have also identified with these approval drugs, we found that they seem to be relatively antagonistic combinations. For example, low concentrations of wide-range. Not only at the highest concentration but also at the cytarabine or topotecan have little effect on proliferation as single lower concentrations, eribulin could synergize with several drugs. agents, but can dramatically antagonize eribulin activity under One challenge with the evaluation of in vitro drug activities is that combination conditions. Indeed, this is not without precedent: patterns of drug exposure may not match what can be achieved in antagonism has been observed for a variety of cancer drug vivo. Limited drug exposure (pulse dosing) could be used to compare combinations in preclinical studies, prompting follow up analyses in vitro results with exposure in a clinical setting. In addition, to investigate both the mechanisms of the synergies as well as the tumor bearing animals could be treated with both drugs to validate effects of drug combination sequencing28 [ -35]. For instance, in combination activities to examine combination drug toxicities. To one extensively studied example, the anthracycline doxorubicin this end, in vivo animal studies are currently in progress; to date, antagonized activity of the vinca alkaloid vincristine in 83% (15/18)

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. 015 Rickles, et al. (2015) of hematopoietic cell lines tested. This antagonism was reproduced 11. Waller CF, Vynnychenko I, Bondarenko I, Shparyk Y, Hodge JP, et al. (2015) in vivo in a xenograft model and was also observed in 34% (12/35) of An Open-Label, Multicenter, Randomized Phase Ib/II Study of Eribulin Mesylate Administered in Combination With Pemetrexed Versus Pemetrexed childhood leukemia cells simultaneously treated with both drugs ex Alone as Second-Line Therapy in Patients With Advanced Nonsquamous vivo [35]. Moreover, combination activity was shown to be sequence Non-Small-Cell Lung Cancer. Clin Lung Cancer 16: 92-99. dependent: if cells are exposed to the anthracycline first, antagonism 12. Schöffski P, Maki RG, Italiano A, Gelderblom H, Grignani G, et al. (2015) is observed, but if exposure to vinca alkaloid precedes anthracycline, Randomized, open-label, multicenter, phase III study of eribulin versus the combination result is beneficial. A p53-dependent mechanism for dacarbazine in patients (pts) with leiomyosarcoma (LMS) and adipocytic this antagonism was proposed, where anthracycline effects stabilize sarcoma (ADI). J Clin Oncol 33: LBA10502. anti-apoptotic BCL2 family members, thus reducing cytotoxic effects 13. Barrentina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, et al. of the vinca alkaloid. With respect to our current study, the eribulin (2012) The Cancer Cell Line Encyclopedia enables predictive modelling of antagonisms observed with 17-DMAG, cytarabine, topotecan and anticancer drug sensitivity. Nature 483: 603-607. gemcitabine do not necessarily require that p53 be functional as was 14. Saiki AY, Caenepeel S, Yu D, Lofgren JA, Osgood T, et al. (2014) MDM2 the case in the preceding example, but further study will be required to antagonists synergize broadly and robustly with compounds targeting fundamental oncogenic signaling pathways. Oncotarget 5: 2030-2043. determine this. Indeed, the mechanisms by which these compounds antagonize eribulin may overlap or be entirely unrelated. 15. Lehar J, Zimmermann GR, Krueger AS, Molnar RA, Ledell JY, et al. (2007) Chemical combination effects predict connectivity in biological systems. Mol In conclusion, we have identified promising preclinical in Syst Biol 3:80 vitro synergy combination partners with eribulin, with compound 16. Lehar J, Krueger AS, Avery W, Heilbut AM, Johansen LM, et al. (2009) mechanistic redundancy helping to validate particular targets and Synergistic drug combinations tend to improve therapeutically relevant pathways as important for selective, synergistic tumor cell killing. selectivity. Nat Biotechnol 27: 659-666. 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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. 017