Richard J Rickles1, Junji Matsui3, Ping Zhu1, Yasuhiro Funahashi2,3, Jill M Grenier1, Janine Steiger1, Nanding Zhao2, Bruce A Littlefield2, Kenichi Nomoto2,3 and Toshimitsu Uenaka2*
1Horizon Discovery Inc., MA, Japan
2Eisai Inc., MA, Japan
3Eisai Co., Ltd., Japan
Received: 17 October, 2015; Accepted: 03 November, 2015; Published: 04 November, 2015
Toshimitsu Uenaka, Ph.D., Executive Director, Production Creation Headquarter, Oncology & Antibody Drug Strategy, CINO (Chief Innovation Officer), E-mail:
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.
© 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.
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.
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 [11. Donoghue M, Lemery SJ, Yuan W, He K, Sridhara R, et al. (2012) Eribulin Mesylate for the Treatment of Patients with Refractory Metastatic Breast Cancer: Use of a "Physician's Choice" Control Arm in a Randomized Approval Trial. Clin Cancer Res 18: 1496-505.,22. Shetty N, Gupta S (2014) Eribulin drug review. South Asian J Cancer 3: 57-59.]. 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 [33. 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.].
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 [44. Towle MJ, Salvato KA, Budrow J, Wels BF, Kuznetsov G, et al. (2001) In vitro and in vivo anticancer activities of synthetic macrocyclic ketone analogs of Halichondrin B. Cancer Res 61: 1013-1021.-99. Polastro L, Aftimos PG, Awada A (2014) Eribulin mesylate in the management of metastatic breast cancer and other solid cancers: a drug review. Expert Rev Anticancer Ther 14: 649-665.]. Although eribulin failed to show meaningful activities in clinical trials of head and neck, pancreatic and advanced non-small cell lung cancer [1010. Scarpace SL (2012) Eribulin mesylate (E7389): review of efficacy and tolerability in breast, pancreatic, head and neck, and non-small cell lung cancer. Clin Ther 34: 1467-1473.,1111. Waller CF, Vynnychenko I, Bondarenko I, Shparyk Y, Hodge JP, et al. (2015) An Open-Label, Multicenter, Randomized Phase Ib/II Study of Eribulin Mesylate Administered in Combination With Pemetrexed Versus Pemetrexed Alone as Second-Line Therapy in Patients With Advanced Nonsquamous Non-Small-Cell Lung Cancer. Clin Lung Cancer 16: 92-99.], improvements in overall survival by eribulin were reported in a Phase 3 trial in advanced soft tissue sarcoma compared with dacarbazine [1212. Schöffski P, Maki RG, Italiano A, Gelderblom H, Grignani G, et al. (2015) Randomized, open-label, multicenter, phase III study of eribulin versus dacarbazine in patients (pts) with leiomyosarcoma (LMS) and adipocytic sarcoma (ADI). J Clin Oncol 33: LBA10502.], 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 anti-cancer agents. We therefore have performed an in vitro study of eribulin combined with 34 anticancer agents in 25 different tumor cell lines of various types, through use of a combination high throughput screening (cHTS) platform. Our goals were to identify drugs that might be paired with eribulin to increase clinical efficacy in metastatic breast cancer, to identify drugs that convert eribulin non-responders to responders, and to identify new therapeutic indications for eribulin utilization. Several compelling synergy effects with approved and emerging drugs were observed. The preclinical data provides insights about future clinical development strategies.
Materials and Methods
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 [1313. Barrentina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, et al. (2012) The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483: 603-607.]. 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 Toronto Research Chemicals. Cell proliferation was measured by ATP Lite 1step Luminescence Assay System (Perkin Elmer).
Cells were thawed and grown in culture media according to vendor's recommendations. Detailed methods on the combination assay were reported in [1414. Saiki AY, Caenepeel S, Yu D, Lofgren JA, Osgood T, et al. (2014) MDM2 antagonists synergize broadly and robustly with compounds targeting fundamental oncogenic signaling pathways. Oncotarget 5: 2030-2043.]. Combinations were analyzed using Synergy Score and Loewe Volume Score, as described in [1515. Lehar J, Zimmermann GR, Krueger AS, Molnar RA, Ledell JY, et al. (2007) Chemical combination effects predict connectivity in biological systems. Mol Syst Biol 3:80,1616. Lehar J, Krueger AS, Avery W, Heilbut AM, Johansen LM, et al. (2009) Synergistic drug combinations tend to improve therapeutically relevant selectivity. Nat Biotechnol 27: 659-666.] 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:
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 [1313. Barrentina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, et al. (2012) The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483: 603-607.,1414. Saiki AY, Caenepeel S, Yu D, Lofgren JA, Osgood T, et al. (2014) MDM2 antagonists synergize broadly and robustly with compounds targeting fundamental oncogenic signaling pathways. Oncotarget 5: 2030-2043.]. The Synergy Score equation integrates the experimentally-observed activity volume at each point in the matrix in excess of a model surface numerically derived from the activity of the component agents using the Loewe model for additivity. Additional terms in the Synergy Score equation are used to normalize for various dilution factors used for individual agents and to allow for comparison of synergy scores across an entire experiment.
Loewe Volume is an additional combination model score used to assess the overall magnitude of the combination interaction in excess of the Loewe additivity model. Loewe Volume is particularly useful when distinguishing synergistic increases in a phenotypic activity (positive Loewe Volume) versus synergistic antagonisms (negative Loewe Volume). Please see reference 13 for more detail.
Self-cross based combination screening analysis
In order to objectively establish hit criteria for the combination screen analysis, twelve compounds were selected to be self-crossed across the twenty-five cell line panel as a means to empirically determine a baseline additive, non-synergistic response. The identity of the twelve self-cross compounds was determined by selecting compounds with a variety of maximum response values and single agent dose response steepness. Those drug combinations which yielded effect levels that statistically superseded those baseline additivity values were considered synergistic. The Synergy Score measure was used for the self-cross analysis. Synergy Scores of self-crosses are expected to be additive by definition and, therefore, maintain a synergy score of zero. However, while some self-cross Synergy Scores are near zero, many are greater suggesting that experimental noise or non-optimal curve fitting of the single agent dose responses are contributing to the slight perturbations in the score. Given the potential differences in cell line sensitivity to the eribulin combination activities, we chose to use a cell-line centric strategy for the self-cross based combination screen analysis, focusing on self-cross behavior in individual cell lines versus global review of the cell line panel activity. Combinations where the Synergy Score is greater than the mean self-cross plus two standard deviations (2σ's) or three standard deviations (3 σ's) can be considered candidate synergies at the 95% and 99% confidence levels, respectively.
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 [1717. Jimeno A (2009) Eribulin: Rediscovering tubulin as an anticancer agent. Clin Cancer Res 15: 3903-3905.]. 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 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.