PROJECT SUMMARY Our laboratory has a long-standing interest in non-Pgp mediated mechanisms of drug resistance, having established several cell line models of resistance focusing on the ABC half-transporter ABCG2. We successfully cloned ABCG2 from a mitoxantrone-resistant colon cancer cell line, that exhibited an ATP-dependent reduction in drug accumulation. Comprising 6 transmembrane domains and a single ATP binding domain, the gene encodes a half-transporter molecule and dimerization is required for activity. Overexpression of ABCG2 renders cells resistant to mitoxantrone and to the camptothecins, topotecan and SN-38 (the active metabolite of irinotecan) in vitro. A large number of both substrates and inhibitors of ABCG2 have been discovered, and the variety of both substrates and inhibitors rivals that described for P-glycoprotein. Since a role for drug transporters in mediating cancer drug resistance has never been defined, our group has studied other aspects of targeting this protein. We have studied a variant protein that may be associated with increased exposure to ABCG2 substrates, and particularly selected carcinogens. There is also a role for ABCG2 in limiting the oral absorption of pharmacologic agents and in limiting the brain uptake through expression in the blood brain barrier (See also project #1). Collectively, this work is best characterized as an effort to both understand and exploit ABCG2 as a therapeutic target to improve anticancer therapy. We have worked on structure and function relationships in the protein. We and others reported impaired transport in cells bearing a single nucleotide polymorphism at amino acid 141 that changes glutamine to lysine. Several lines of evidence suggest that this polymorphic variant could be associated with increased exposure to drugs that are substrates of ABCG2, or to a subset of carcinogens that are also known to be substrates of ABCG2, including those in tobacco smoke. This variant was first linked with an increased susceptibility to gout due to reduced excretion of urate and a corresponding increase in blood levels of urate. Similarly, gastrointestinal absorption has also been related to ABCG2 expression in the intestinal epithelium, and one implication of this work is that the Q141K SNP could be associated with increased absorption of substrate drugs such as topotecan, imatinib, or irinotecan. We have focused on understanding the trafficking and function of this variant that results from misfolding and targeting to the aggresome. We have also found that the protein that is targeted to the cell surface has impaired transport activity, and studies are underway in collaboration with Dr. Suresh Ambudkar to understand this abnormal function. Based on our earlier work with the dimerization motif in ABCG2, we have studied the pharmacologic rescue of ABCG2 through the addition of substrates. In fact, the best mediator of rescue was romidepsin, apparently due to at least three mechanisms: increased RNA expression, reduced transfer to the aggressome, and improved surface localization due to improved folding. ABCG2 is expressed in the endothelial cells in the brain and another important role for the protein is in protection of the CNS as a component of the blood-brain barrier. An implication of this finding is that compounds that circumvent ABCG2 and thus, the blood-brain barrier, could have increased efficacy in treating or preventing CNS metastases. The importance of localization of ABCG2 in the blood brain barrier will increase as we recognize ABCG2 substrates in new agents such as small molecule tyrosine kinase inhibitors. In collaboration with Dr. Michael Dean and the Molecular Targets Development Program, led by Dr. James McMahon, ABCG2-overexpressing cells have been used to screen for inhibitors of ABCG2. This is an important undertaking since the potential ability to modulate oral drug absorption and CNS uptake will be important whether or not ABCG2 proves important in oncologic drug resistance. Dr. Curtis Henrich and Kirk Gustafson, of the Molecular Targets Development Program, has identified a number of hits that we have now confirmed as ABCG2 inhibitors, most interesting of which is a family of botryllamide compounds. These compounds were evaluated in secondary screens in our laboratory and in that of Dr. Suresh Ambudkar, to prioritize for further preclinical development. Our plan is to take these compounds into animal studies in collaboration with Dr. William Figg's group. In particular, we hope to assess whether any of these inhibitors will increase CNS accumulation of ABCG2 and Pgp drug substrates.