Genotype-Selective Combination Therapies for Melanoma Identified by High-Throughput Drug Screening

Departments of 1Pathology and 2Dermatology, Yale University School of Medicine, New Haven, Connecticut.
Cancer Discovery (Impact Factor: 19.45). 12/2012; 3(1). DOI: 10.1158/2159-8290.CD-12-0408
Source: PubMed


Resistance and partial responses to targeted monotherapy are major obstacles in cancer treatment. Systematic approaches to identify efficacious drug combinations for cancer are not well established, especially in the context of genotype. To address this, we have tested pairwise combinations of an array of small-molecule inhibitors on early-passage melanoma cultures using combinatorial drug screening. Results reveal several inhibitor combinations effective for melanomas with activating RAS or BRAF mutations, including mutant BRAF melanomas with intrinsic or acquired resistance to vemurafenib. Inhibition of both EGF receptor and AKT sensitized treatment-resistant BRAF mutant melanoma cultures to vemurafenib. Melanomas with RAS mutations were more resistant to combination therapies relative to BRAF mutants, but were sensitive to combinations of statins and cyclin-dependent kinase inhibitors in vitro and in vivo. These results show the use of combinatorial drug screening for discovering unique treatment regimens that overcome resistance phenotypes of mutant BRAF- and RAS-driven melanomas.

We have used drug combinatorial screening to identify effective combinations for mutant BRAF melanomas, including those resistant to vemurafenib, and mutant RAS melanomas that are resistant to many therapies. Mechanisms governing the interactions of the drug combinations are proposed, and in vivo xenografts show the enhanced benefit and tolerability of a mutant RAS -selective combination, which is currently lacking in the clinic.

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    • "The application of phenotypic drug combination screening is exemplified by dose-ratio matrix testing multiple pairwise combinations across cell based assays enabling analysis of synergy, additive, and antagonistic effects across diverse chemical libraries, annotated compound libraries and approved drug sets (Zimmermann et al., 2007). Recent examples of dedicated drug combination screening campaigns using a variety of phenotypic assays and distinct endpoints have been published (Axelrod et al., 2013; Cubitt et al., 2013; Du et al., 2013; Held et al., 2013; Schmidt et al., 2013; Li et al., 2014). Such phenotypic screens have identified novel synergistic combinations such as: Lapatanib (EGFR and Her2 inhibitor) combined with the multi-targeted inhibitor Ro31-8220 (Axelrod et al., 2013); Lapatanib combined with MK2206 (Akt inhibitor) (Held et al., 2013) and Rapamycin (mTOR) combined with Sunitinib (multi-targeted kinase inhibitor) (Li et al., 2014). "
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    ABSTRACT: Advances in target-based drug discovery strategies have enabled drug discovery groups in academia and industry to become very effective at generating molecules that are potent and selective against single targets. However, it has become apparent from disappointing results in recent clinical trials that a major challenge to the development of successful targeted therapies for treating complex multifactorial diseases is overcoming heterogeneity in target mechanism among patients and inherent or acquired drug resistance. Consequently, reductionist target directed drug-discovery approaches are not appropriately tailored toward identifying and optimizing multi-targeted therapeutics or rational drug combinations for complex disease. In this article, we describe the application of emerging high-content phenotypic profiling and analysis tools to support robust evaluation of drug combination performance following dose-ratio matrix screening. We further describe how the incorporation of high-throughput reverse phase protein microarrays with phenotypic screening can provide rational drug combination hypotheses but also confirm the mechanism-of-action of novel drug combinations, to facilitate future preclinical and clinical development strategies.
    Frontiers in Pharmacology 05/2014; 5:118. DOI:10.3389/fphar.2014.00118 · 3.80 Impact Factor
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    • "Dasatinib, an inhibitor of BCR-Abl kinase and SRC kinase, exhibited synergistic growth inhibition with afatinib in NSCLC lines expressing L858R/T790M or the combination of del E746-A750 with a deletion of exon 9 in PTEN (Chang and Wang 2012). In BRAF mutant melanoma cell lines, afatinib also had a little effect as a single agent but became a more potent growth inhibitor in the presence of Akt inhibitors (GSK690693 and MK-2206) (Held et al. 2013). Pfeifer and colleagues have systematically explored the compound combination PI-103 (dual PI3K/mTOR inhibitor) with afatinib on a panel of 65 NSCLC cell lines (Pfeifer et al. 2010). "
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    ABSTRACT: Afatinib (also known as BIBW 2992) has recently been approved in several countries for the treatment of a distinct type of epidermal growth factor receptor (EGFR)-mutated non-small cell lung cancer. This manuscript comprehensively reviews the preclinical data on afatinib, an irreversible inhibitor of the tyrosine kinase activity of members of the epidermal growth factor receptor family (ErbB) including EGFR, HER2 and ErbB4. Afatinib covalently binds to cysteine 797 of the EGFR and the corresponding cysteines 805 and 803 in HER2 and ErbB4, respectively. Such covalent binding irreversibly inhibits the tyrosine kinase activity of these receptors, resulting in reduced auto- and transphosphorylation within the ErbB dimers and inhibition of important steps in the signal transduction of all ErbB receptor family members. Afatinib inhibits cellular growth and induces apoptosis in a wide range of cells representative for non-small cell lung cancer, breast cancer, pancreatic cancer, colorectal cancer, head and neck squamous cell cancer and several other cancer types exhibiting abnormalities of the ErbB network. This translates into tumour shrinkage in a variety of in vivo rodent models of such cancers. Afatinib retains inhibitory effects on signal transduction and in vitro and in vivo cancer cell growth in tumours resistant to reversible EGFR inhibitors, such as those exhibiting the T790M mutations. Several combination treatments have been explored to prevent and/or overcome development of resistance to afatinib, the most promising being those with EGFR- or HER2-targeted antibodies, other tyrosine kinase inhibitors or inhibitors of downstream signalling molecules.
    Archiv für Experimentelle Pathologie und Pharmakologie 03/2014; 387(6). DOI:10.1007/s00210-014-0967-3 · 2.47 Impact Factor
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    • "Many combination therapies are used to treat metastatic breast cancer [34]. Several inhibitor combinations effective for melanomas with activating RAS or BRAF mutations were recently discovered [35]. Combination therapy of α-galactosylceramide and 5-fluorouracil showed antitumor effect in mice and suggests a new therapy mode against metastatic liver cancer [36]. "
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    ABSTRACT: Epigenetic therapy reverting aberrant acetylation or methylation offers the possibility to target preferentially tumor cells and to preserve normal cells. Combination epigenetic therapy may further improve the effect of individual drugs. We investigated combined action of demethylating agent decitabine and histone deacetylase inhibitor SAHA (Vorinostat) on different leukemic cell lines in comparison with peripheral blood lymphocytes. Large decrease of viability, as well as huge p21WAF1 induction, reactive oxygen species formation, and apoptotic features due to combined decitabine and SAHA action were detected in leukemic cell lines irrespective of their p53 status, while essentially no effect was observed in response to the combined drug action in normal peripheral blood lymphocytes of healthy donors. p53-dependent apoptotic pathway was demonstrated to participate in the wtp53 CML-T1 leukemic cell line response, while significant influence of reactive oxygen species on viability decrease has been detected in p53-null HL-60 cell line.
    08/2013; 2013(2):659254. DOI:10.1155/2013/659254
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