Characterization of the Q141K Single Nucleotide Polymorphism We and others have reported on the impaired functionality of the Q141K SNP in ABCG2, with most investigators identifying lower cell surface levels as a mechanism of impairment and our data suggesting an additional functional impairment after correction for surface expression. The clinical impact of this SNP has been confirmed, with higher plasma levels of orally administered drugs, including oral topotecan and diflomotecan, demonstrated in patients with the Q141K SNP (93,94). Reduced uric acid excretion linked to this SNP has been convincingly linked to gout. Another possibility is that the Q141K SNP results in a misfolded protein. As noted above, often ABCG2 proteins that are not properly folded are degraded by the ERAD pathway. Ishikawa observed that the Q141K SNP leads to protein recognition by ERAD. Based on work with the cystic fibrosis transporter, we postulated that certain substrates could rescue Q141K ABCG2 from degradation. Indeed, we were able to show that both colchicine and romidepsin increased Q141K ABCG2 at the cell surface. This strategy could potentially be used to mediate increased efflux of inhaled or ingested carcinogens via increased expression of Q141K ABCG2 in a prevention scheme. One of the remarkable observations regarding Q141K is that its pharmacologic and physiologic impact has been so readily detected in the clinic. This contrasts with variants of P-glycoprotein around which there has been much controversy. Our laboratory studies suggested that even when the protein reached the cell surface the transport efficiency is reduced. We have collaborated with Dr. Suresh Ambudkar and Dr. Suneet Shukla to study the biochemistry of this polymorphism. Data based on photo-crosslinking with 125I-iodoarylazidoprazosin suggest that the Q141K SNP does not affect drug binding. Only slight differences in ATP hydrolysis were observed, and these were not felt to be physiologically significant. Measurement of half-life is ongoing, but a reduction would not be surprising, and we hypothesize will be normalized by the same agents we used to show improved trafficking. A finding that neither functional abnormalities can be detected, nor a reduced half-life would indicate that the defect in Q141K lies entirely in level of expression at the cell surface. Development of Novel, Potent ABC Transporter Inhibitors for Clinical Use in the CNS While expression of ABCG2 in the gut and brain endothelium serves a protective role in normal physiology, it can be detrimental during cancer treatment. Expression of ABCG2 alone or in combination with Pgp has been shown to limit oral bioavailability and brain penetration of topotecan as well as several targeted therapies including imatinib, dasatinib, lapatinib, sorafenib and erlotinib in mice. Since lung and breast cancers often metastasize to the brain, there is the possibility ABCG2 limits brain penetration of these therapies, rendering them less effective. Thus, a transport inhibitor that increased delivery of targeted therapies across the blood-brain barrier could be used to prevent or treat brain metastases. Interestingly, in mouse models, when either Pgp or ABCG2 alone is deleted, there is often a modest impact on brain penetration - but there is a considerable impact when both are deleted. Polli and colleagues found that the greatest increase in brain penetration of lapatinib occurred in mice lacking both Abcg2 and Pgp, raising the possibility that a dual inhibitor of Pgp and ABCG2 may be necessary. Administering the dual ABCG2/ Pgp inhibitor, elacridar (GF120918), with topotecan resulted in complete oral bioavailability and decreased inter-patient variability. Studies in mice have demonstrated that increased brain penetration of imatinib and its active metabolite, CGP74588, can be achieved by co-administration of elacridar with imatinib. Similar findings have been reported for dasatinib. We have previously shown that tariquidar, the Pgp modulator we have studied in clinical trials, also inhibits ABCG2. Currently, the majority of available inhibitors lack potency with respect to ABCG2 or have toxic effects. To develop potent ABC transporter inhibitors for clinical applications, we collaborated with Drs. James McMahon and Curtis Henrich of the Molecular Targets Laboratory to develop a high-throughput screen for novel inhibitors of ABCG2. The screen was based on accumulation of the ABCG2 specific substrate pheophorbide a in ABCG2-overexpressing NCI-H460 MX20 cells. The NCI-DTP natural and synthetic compound library (7,325 compounds) as well as the NCI natural products extracts library (91,000 compounds) were screened. The natural and synthetic compound library yielded 5 lead compounds, of which NSC11668 was selected for further study. The natural product extracts library yielded a new class of ABCG2 interacting compounds, the botryllamides, from which 2 were selected for further study, one of which is an inhibitor of both Pgp and ABCG2. Assay of the ability of the botryllamides to improve lapatinib uptake is ongoing in preclinical in vivo models of CNS uptake and oral drug bioavailability in the laboratory of Dr. William Figg. Our goal is to follow this study, if positive, with proof of concept pre-clinical studies involving brain metastasis models.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010622-12
Application #
9153614
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
12
Fiscal Year
2015
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Zhang, Hui; Kathawala, Rishil J; Wang, Yi-Jun et al. (2014) Linsitinib (OSI-906) antagonizes ATP-binding cassette subfamily G member 2 and subfamily C member 10-mediated drug resistance. Int J Biochem Cell Biol 51:111-9
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