Cisplatin and carboplatin (platinum drugs) are among the most successful antitumor drugs and are used to treat testicular, breast, ovarian, bladder, neck and lung cancer. As with many antitumor agents, the efficacy of treatment can vary greatly from patient to patient and the occurrence of resistance is a significant problem. The therapeutic effect of the platinums is based on the formation of different types of DNA adducts, primarily intra- and interstrand crosslinks, by the reaction with two purine bases on one or two complementary strands of DNA. The resistance to platinum treatment occurs by a the up- or down-regulation of several processes, including metal transport in and out of the cell, intracellular metabolism and the removal of the cisplatin DNA adducts by DNA repair pathways. The primary hypothesis of the work proposed in this application is that by developing a monoclonal antibodies with specificity for the individual platinum DNA adducts in the genomes of cells we will be able to 1) determine which platinum adduct is the most therapeutically relevant one, 2) understand the repair mechanisms responsible for resistance, 3) test the sensitivity or resistance to platinum in tumor biopsies and 4) to select patients that are most likely to benefit from therapy and sparing those that are likely to be resistant the severe side effect associated with this type of treatment. In this R21 application, we propose to synthesize oligonucleotides containing the 1,2-GG-, 1,2-AG-, 1,3-GNG-intrastrand and 1,2-GC-interstrand platinum crosslinks, couple them to the KLH carrier protein and use them to immunize mice to generate monoclonal antibodies that recognize the individual adducts with high specificity. These monoclonal antibodies will be then further validated in cell lines with specific DNA repair defects. We predict that these reagents will enable us to show that platinum intrastrand crosslinks persist in cells with defects in the nucleotide excision repair (NER) pathway, while interstrand crosslinks will persist in cells with defects interstrand crosslink (ICL) repair pathwa. We will then measure platinum levels in breast and ovarian cancer cell lines of the NCI-60 collection, which have been characterized for drug sensitivity or resistance. We will determine whether the levels of a specific adduct or all adduct are elevated in platinum-responsive cell lines. Conversely, we will test whether a specific adduct is absent in resistant cell lines. We expect that upon completion of the studies proposed here we will have developed a set of unique reagents that will be of tremendous use for the research and medical community to determine which platinum adducts are clinically most relevant and to work toward developing a robust method to predict clinical outcomes of platinum treatments.
Cisplatin and carboplatin are among the most commonly used and successful cancer chemotherapeutic drugs, but resistance of tumors to treatment and drug toxicity associated with the platinum drugs are problematic. The work proposed here aims to generate reagents that can be used to detect the levels of four different cytotoxic platinum adducts in the DNA of tumor cells. The exact measurement of platinum adduct levels will make it possible to determine which tumors are likely to be resistant to treatment. This approach should eventually make it possible to make informed decisions about which patients are likely to benefit from treatment with platinum drugs, with profound impact on the treatment of testicular, lung, bladder, ovarian, neck and lung cancer.
