Two topoisomerase I (topo I) poisons, topotecan (TPT) and irinotecan (CPT-11), are approved for use in the U.S.; and additional topo I poisons are currently undergoing clinical testing. Because clinical responses to these agents are highly variable, studies supported by this grant have investigated 1) mechanisms of resistance to this class of agents in clinical specimens and preclinical models and 2) effects of combining topo I poisons with other agents. In clinical specimens, our studies have shown that a) response of gastric and gastroesophageal junction cancer to CPT-11 does not correlate with staining for topo I, Ki67 (a marker of proliferation), or Bcl-2 family members; b) response of ovarian cancer to TPT tentatively correlates with topo I content, although the strength of this conclusion is limited by small sample size; and c) response of acute leukemia to TPT-containing therapy correlates with low Bcl-2 content in two separate trials. In preclinical models, we have identified a role for signaling by the ATR/Chk1 pathway in sensitivity to topo I poisons. Further studies in preclinical models have allowed us to map three different phosphorylation sites on topo I, one that is induced by various stimuli and two that are not, placing us in a unique position to examine the role of various topo I Sation events. Finally, in preclinical studies examining drug combinations, we showed that topo I poisons synergize with the epidermal growth factor receptor tyrosine kinase inhibitor OSI-774 (based on the ability of OSI-774 to inhibit efflux of SN-38 by the ABCG2 transporter) and with the heat shock protein 90 (Hsp90) inhibitor 17-allylamino-17-demethoxy- geldanamycin. These observations have led to an ongoing trial of the CPT-11/OSI-774 combination and a planned trial of a CPT-11/17-dimethylaminoethylamino-17-demethoxygeldanamycin combination. To build on these results, we now propose to 1) investigate the mechanistic basis for ATR/Chk1 pathway activation by topo I poisons in greater detail, 2) determine the physiological and pharmacological consequences of topo I phosphorylation at each site, and 3) examine the effect of combining topo I poisons with inhibitors of pathways implicated in resistance in our recent studies. Collectively, these studies are designed to provide new insight into the mechanism of action of topo I poisons, and identify strategies for enhancing their efficacy. ? ? ? ?
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