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An in vitro screening to identify drug-resistant mutations for target-directed chemotherapeutic agents.

Divisions of Pathology, Hematology and Cancer Biology, Cancer and Blood Disease Institute, Cincinnati Children's Hospital and Medical Center, Cincinnati, OH, USA, .
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2012; 928:175-84. DOI: 10.1007/978-1-62703-008-3_14
Source: PubMed

ABSTRACT The discovery of oncogenes and tumor suppressors as a driver of cancer development has triggered the development of target-specific small molecule anticancer compounds. As exemplified by Imatinib (Gleevec), a specific inhibitor of the Chronic Myeloid Leukemia-associated BCR/ABL kinase, these agents promise impressive activity in clinical trials, with low levels of clinical toxicity. However, such therapy is susceptible to the emergence of drug resistance mainly due to amino acid substitutions in the target protein. Defining the spectrum of such mutations is important for patient monitoring and the design of next-generation inhibitors. Using Imatinib and BCR/ABL as a paradigm for a drug-target pair, we reported a retroviral vector-based screening strategy to identify the spectrum of resistance-conferring mutations, which has helped in designing the next-generation BCR/ABL inhibitors such as Nilotinib, Dasatinib, and Ponatinib. Here we provide a detailed methodology for the screen, which can be generally applied to any drug-target pair.

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    ABSTRACT: Acquired resistance to targeted kinase inhibitors is a well-documented clinical problem that is potentially fatal for patients to whom a suitable back-up is not available. However, protein kinase alleles that promote resistance to inhibitors can be exploited experimentally as gold-standards for "on"- and "off"-target validation strategies and constitute a powerful resource for assessing the ability of new or combined therapies to override resistance. Clinical resistance to kinase inhibitors is an evident in all tyrosine kinase-driven malignancies, where high rates of mutation drive tumor evolution toward the insidious drug-resistant (DR) state through a variety of mechanisms. Unfortunately, this problem is likely to intensify in the future as the number of target kinases, approved inhibitors, and clinical indications increase. To empower the analysis of resistance in kinases, we have validated a bioinformatic, structural, and cellular workflow for designing and evaluating resistance at key mutational hotspots among kinome members. In this chapter, we discuss how mutation of amino acids in the gatekeeper and hinge-loop regions (collectively termed the "resistance tetrad") and the DFG motif represent an effective approach for generating panels of DR kinase alleles for chemical genetics and biological target validation.

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May 22, 2014