Targeted therapies are used depending on the cancer genotype or stage of disease. Sorafenib, erlotinib and lapatinib are small molecule tyrosine kinase.
miR-34 Mimics Synergize with Small Molecule Inhibitors Targeting the EGFR and Raf Kinase Pathways Jane Zhao, Kevin Kelnar, Christopher Daige, Jason Wiggins, Leslie Priddy, Terri Muenzer, David Brown and Andreas G. Bader Mirna Therapeutics, Inc., 2150 Woodward #100, Austin, Texas 78744
ABSTRACT Sorafenib (Nexavar®), erlotinib (Tarceva®) and lapatinib (Tykerb®) are FDAapproved therapies for patients with hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), and metastatic breast cancer, respectively. Sorafenib is a multi-kinase inhibitor targeting the Raf/Mek/Erk pathway, erlotinib is a tyrosine kinase inhibitor antagonizing epidermal growth factor receptor (EGFR), and lapatinib is a dual tyrosine kinase inhibitor interrupting the HER2 and EGFR pathways. Due to their selective inhibitory action and observed toxicities, current treatment options for liver, lung and breast cancer are limited and many cancers develop resistance. Naturally occurring tumor suppressor microRNAs inhibit tumor growth by regulating multiple oncogenes at once and, therefore, microRNA mimics, which are copies of the naturally occurring microRNAs, may be used in combination with the respective standard of care drugs to bring this tumor suppressor activity back into tumor cells and augment drug sensitivity. Here, we investigated the relationship of a mimic of the tumor suppressor microRNA miR-34 in combination with the small molecule inhibitors and determined the therapeutic activity of the combination in cancer cells. Data derived from isobolograms, combination index plots and curve shift analyses indicate synergy in all cancer cell lines tested. Synergy was observed at multiple microRNA and drug ratios and at drug concentrations that induce 50% or greater cancer cell inhibition.
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S ynergy between erlotinib and miR-34 mimics was observed across a range of different dose levels and drug ratios in all NSCLC and HCC cancer cells tested. Maximal synergy was detected at dosages that provide a high level of cancer cell inhibition beyond the one that is induced by the single agents alone and, thus, is potentially of clinical relevance. T he combination of lapatinib and miR-34 mimics produced moderate synergistic effects in breast cancer cells. The combination of sorafenib and miR-34 mimics produced synergistic effects in cultured HCC cells and as well as in orthotopic liver tumor xenografts.
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INTRODUCTION Targeted therapies are used depending on the cancer genotype or stage of disease. Sorafenib, erlotinib and lapatinib are small molecule tyrosine kinase inhibitors (TKIs) of the Raf kinase, EGFR and HER2 oncogenic signaling pathways, respectively. They function as competitive inhibitors by binding to the ATPbinding sites of the targeted kinases. Clinical trials revealed that therapeutic responses to these kinase inhibitors occurred in a limited fraction of cancer patients due to either primary or acquired resistance.1-6 Because miRNAs can regulate multiple oncogenic pathways at once, we believe that synthetic mimics of tumor suppressor miRNAs may be used to overcome drug resistance mechanisms and to create more effective therapies.7-8 There are several studies demonstrating how miR-34 mimics can sensitize cancer cells to conventional cytotoxic chemotherapies.12-14 However, the combination of tumor suppressor miRNAs with targeted therapies is a less well studied approach. Here, we examined whether miR‑34 mimics can sensitize cancer cells to TKIs.
T he combination of erlotinib + miR-34 mimics produced strong synergistic effects in all NSCLC and HCC cancer cells tested.
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Figure 1. Erlotinib and miR-34 mimics synergize in NSCLC. (A) Combination index plot showing synergy at high levels of cancer cell inhibition in four NSCLC cell lines. (B) Dose response-curves of the combination and single agents. Drug concentrations are normalized to IC50 equivalents. The combination shows increased maximal inhibitory activity. (C) Isobolograms illustrating reduced dose requirements to achieve 50% and 80% cancer cell inhibition (Fa). (D, top left) IC50 of erlotinib in sensitive HCC827 lung cancer cells. (D, bottom left) Micrograph of resistant HCC827-res cells in the presence of high erlotinib concentrations (IC90 of HCC827 cells). (D, right) Dose response-curves of erlotinib in the presence of a weak, constant miR-34 or miR-NC concentration (0.3 nM).
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METHOD The combinatorial effects were evaluated in a number of cancer cell lines. Cancer cells completely resistant to TKIs were used in a “fixed concentration” method whereby cells were treated with a fixed, weak concentration of both miR-34 mimics and TKIs in a serial dilution. Drug enhancements were determined by a visual inspection of the erlotinib dose-response curve and a decrease of the IC50 value in the presence of miR-34. Cancer cells partially resistant to TKIs were used in a “fixed ratio” method whereby multiples of drug concentrations were combined such that the ratio of the two agents remained constant. Synergistic, additive or antagonistic effects were determined according to Chou-Talalay’s algorithm and visualized in combination index (CI) plots, isobolograms and curve shift analyses.9-11 Effects of liposomal miR-34 (MRX34) in combination with sorafenib were further evaluated in an orthotopic mouse model inoculated with Hep3B liver cancer cells. Tumor growth was measured by serum alpha-fetoprotein (AFP) levels.
11. Zhao L, Au JL Wientjes MG (2010) Comparison of methods for evaluating drug-drug interaction. Front Biosci (Elite Ed) 2: 241-9. 12. Ji Q, Hao X, Meng Y, Zhang M, Desano J, et al. (2008) Restoration of tumor suppressor miR-34 inhibits human p53-mutant gastric cancer tumorspheres. BMC Cancer 8: 266. 13. Vinall RL, Ripoll AZ, Wang S, Pan CX deVere White RW (2011) MiR-34a chemosensitizes bladder cancer cells to cisplatin treatment regardless of p53-Rb pathway status. Int J Cancer 130: 2526-38. Figure 4. Sorafenib and miR-34 mimics synergize in HCC. (A) Combination index values indicate synergy in three HCC lines. (B) Isobologram predicts reduced dose requirements for miR-34 mimics and sorafenib to achieve 50% cancer cell inhibition. (C) Superior effects of liposomal miR-34 (MRX34) in combination with sorafenib in a mouse model of hepatocellular carcinoma. Three weeks after surgical implantation of Hep3B liver cancer xenografts into mouse livers, mice were treated with MRX34 (0.1 mg/kg, tiw), sorafenib (10 mg/kg, qd), or with the combination. As controls, groups of mice were treated with vehicle (empty liposomes and PO). Tumor growth was measured by serum AFP levels. Figure 2. Erlotinib and miR-34 mimics synergize in HCC. (A) Combination index plot showing synergy at high levels of cancer cell inhibition in four HCC cell lines. (B) Isobolograms illustrating reduced dose requirements to achieve 50% and 80% cancer cell inhibition (Fa) in Hep3B cells.
Figure 3. Effects of lapatinib in combination with miR-34 mimics in breast cancer cells. (A) Combination index plot showing synergy at high levels of cancer cell inhibition in three breast cancer cell lines. (B) Isobolograms illustrating reduced dose requirements to achieve 50% and 80% cancer cell inhibition (Fa) in MDA MB 231 cells.
14. Weeraratne SD, Amani V, Neiss A, Teider N, Scott DK, et al. (2010) miR-34a confers chemosensitivity through modulation of MAGE-A and p53 in medulloblastoma. Neuro Oncol 13: 165-75.
