ABCG2-overexpressing Cell Lines Research Articles

Abstract 360: Methyl-cantharidimide (MCA) can induce apoptosis by activating UNC5B-Netrin-1-DAPK pathway in hepatocellular carcinoma

Abstract In this study, we investigated the underlying mechanism of action of methyl-cantharidimide (MCA), a cantharidin (CTD) analog, as an anticancer drug, in resistant cancer cells overexpressing either ABCB1 or ABCG2 and in cisplatin-resistant cancer cells. The results indicated that: (i) MCA was efficacious in the ABCB1-overexpressing cell line, KB-C2, the ABCG2-overexpressing cell line, NCI-H460/MX20 and in the cisplatin resistant cancer cell lines, KCP-4 and BEL-7404/CP20; (ii) MCA induced apoptosis in both BEL-7404 and BEL-7404/CP20 cancer cells and arrested both BEL-7404 and BEL-7404/CP20 cancer cells in the G0/G1 phase of the cell cycle; (iii) MCA upregulated the expression level of the protein, unc-5 netrin receptor B (UNC5B) in HepG2 and BEL-7404 cancer cells. (iv) MCA can activate UNC5B-Netrin-1-DAPK apoptosis pathway in hepatocellular carcinoma cells. Overall, our results indicated that MCA's efficacy in multiple cancer cell lines is due to the activation of UNC5B-Netrin-1-DAPK apoptosis pathway and cell cycle arrest in the G0/G1 phase. Citation Format: Yi-Dong Li, Yong Mao, Xing-Duo Dong, Zi-Ning Lei, Yuqi Yang, Lizhu Lin, Dong-Hua Yang, Zhe-Sheng Chen. Methyl-cantharidimide (MCA) can induce apoptosis by activating UNC5B-Netrin-1-DAPK pathway in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 360.

BMS-599626, a Highly Selective Pan-HER Kinase Inhibitor, Antagonizes ABCG2-Mediated Drug Resistance

Simple SummaryABC transporters comprise a large group of ATP binding plasma membrane proteins, classified into subfamilies A-G, that transport substrates out of cells to maintain homeostasis. Prolonged exposure to chemotherapeutic drugs leads to increased expression of ABC transporters in cancer cells, resulting in increased efflux and decreased efficacy of anti-neoplastic agents. We found that BMS-599626, at 300 nM, inhibited the function of ABCG2, thereby increasing the efficacy of substrate chemotherapeutic drugs in wild-type as well as mutant ABCG2 overexpressing cells. In addition, BMS-599626 did not alter the expression or intracellular localization of ABCG2 but produced its reversal effect by decreasing efflux and increasing the intracellular accumulation of substrate chemotherapeutic drugs. Finally, BMS-5999626 also inhibited ABCG2 mediated ATP hydrolysis. Overall, our results show that administration of BMS-599626 along with chemotherapeutic drugs can improve the efficacy of chemotherapy in ABC transporter overexpressing cancer cells.Multidrug resistance (MDR) associated with the overexpression of ABC transporters is one of the key causes of chemotherapy failure. Various compounds blocking the function and/or downregulating the expression of these transporters have been developed over the last few decades. However, their potency and toxicity have always been a concern. In this report, we found that BMS-599626 is a highly potent inhibitor of the ABCG2 transporter, inhibiting its efflux function at 300 nM. Our study repositioned BMS-599626, a highly selective pan-HER kinase inhibitor, as a chemosensitizer in ABCG2-overexpressing cell lines. As shown by the cytotoxicity assay results, BMS-599626, at noncytotoxic concentrations, sensitizes ABCG2-overexpressing cells to topotecan and mitoxantrone, two well-known substrates of ABCG2. The results of our radioactive drug accumulation experiment show that the ABCG2-overexpressing cells, treated with BMS-599626, had an increase in the accumulation of substrate chemotherapeutic drugs, as compared to their parental subline cells. Moreover, BMS-599626 did not change the protein expression or cell surface localization of ABCG2 and inhibited its ATPase activity. Our in-silico docking study also supports the interaction of BMS-599626 with the substrate-binding site of ABCG2. Taken together, these results suggest that administration of chemotherapeutic drugs, along with nanomolar concentrations (300 nM) of BMS-599626, may be effective against ABCG2-mediated MDR in clinical settings.

Sitravatinib, a Tyrosine Kinase Inhibitor, Inhibits the Transport Function of ABCG2 and Restores Sensitivity to Chemotherapy-Resistant Cancer Cells in vitro.

Sitravatinib, also called MGCD516 or MG-516, is a broad-spectrum tyrosine kinase inhibitor (TKI) under phase III clinical evaluation. Herein, we explored the activity of sitravatinib toward multidrug resistance (MDR) by emphasizing its inhibitory effect on ATP-binding cassette super-family G member 2 (ABCG2). ABCG2 is a member of ATP-binding cassette (ABC) transporter family and plays a critical role in mediating MDR. Sitravatinb received an outstanding docking score for binding to the human ABCG2 model (PDB code: 6ETI) among thirty screened TKIs. Also, an MTT assay indicated that sitravatinib at 3 μM had the ability to restore the antineoplastic effect of various ABCG2 substrates in both drug-selected and gene-transfected ABCG2-overexpressing cell lines. In further tritium-labeled mitoxantrone transportation study, sitravatinib at 3 μM blocked the efflux function mediated by ABCG2 and as a result, increased the intracellular concentration of anticancer drugs. Interestingly, sitravatinib at 3 μM altered neither protein expression nor subcellular localization of ABCG2. An ATPase assay demonstrated that ATPase activity of ABCG2 was inhibited in a concentration-dependent manner with sitravatinib; thus, the energy source to pump out compounds was interfered. Collectively, the results of this study open new avenues for sitravatinib working as an ABCG2 inhibitor which restores the antineoplastic activity of anticancer drugs known to be ABCG2 substrates.

Benzoyl indoles with metabolic stability as reversal agents for ABCG2-mediated multidrug resistance

Ko143, a potent ABCG2 inhibitor that reverses multidrug resistance in cancer, cannot be used clinically due to its unsuitable metabolic stability. We identified benzoyl indoles as reversal agents that reversed ABCG2-mediated multidrug resistance (MDR), with synthetic tractability and enhanced metabolic stability compared to Ko143. Bisbenzoyl indole 2 and monobenzoyl indole 8 significantly increased the accumulation of mitoxantrone (MX) in ABCG2-overexpressing NCI–H460/MX20 cells, and sensitized NCI–H460/MX20 cells to mitoxantrone. Mechanistic studies were conducted by [3H]-MX accumulation assay, Western blot analysis, immunofluorescence analysis and ABCG2 ATPase assay. The results revealed that the reversal efficacies of compounds 2 and 8 were not due to an alteration in the expression level or localization of ABCG2 in ABCG2-overexpressing cell lines. Instead, compounds 2 and 8 significantly stimulated the ATP hydrolysis of ABCG2 transporter, suggesting that these compounds could be competitive substrates of ABCG2 transporter. Overall, the results of our study indicated that compounds 2 and 8 significantly reversed ABCG2-mediated MDR by blocking the efflux of anticancer drugs.

Olmutinib (BI1482694/HM61713), a Novel Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, Reverses ABCG2-Mediated Multidrug Resistance in Cancer Cells.

The main characteristic of tumor cell resistance is multidrug resistance (MDR). MDR is the principle cause of the decline in clinical efficacy of chemotherapeutic drugs. There are several mechanisms that could cause MDR. Among these, one of the most important mechanisms underlying MDR is the overexpression of adenosine triphosphate (ATP)-binding cassette (ABC) super-family of transporters, which effectively pump out cytotoxic agents and targeted anticancer drugs across the cell membrane. In recent years, studies found that ABC transporters and tyrosine kinase inhibitors (TKIs) interact with each other. TKIs may behave as substrates or inhibitors depending on the expression of specific pumps, drug concentration, their affinity for the transporters and types of co-administered agents. Therefore, we performed in vitro experiments to observe whether olmutinib could reverse MDR in cancer cells overexpressing ABCB1, ABCG2, or ABCC1 transporters. The results showed that olmutinib at 3 μM significantly reversed drug resistance mediated by ABCG2, but not by ABCB1 and ABCC1, by antagonizing the drug efflux function in ABCG2-overexpressing cells. In addition, olmutinib at reversal concentration affected neither the protein expression level nor the localization of ABCG2. The results observed from the accumulation/efflux study of olmutinib showed that olmutinib reversed ABCG2-mediated MDR with an increasing intracellular drug accumulation due to inhibited drug efflux. We also had consistent results with the ATPase assay that olmutinib stimulated ATPase activity of ABCG2 up to 3.5-fold. Additionally, the molecular interaction between olmutinib and ABCG2 was identified by docking simulation. Olmutinib not only interacts directly with ABCG2 but also works as a competitive inhibitor of the transport protein. In conclusion, olmutinib could reverse ABCG2-mediated MDR. The reversal effect of olmutinib on ABCG2-mediated MDR cells is not due to ABCG2 expression or intracellular localization, but rather related to its interaction with ABCG2 protein resulting in drug efflux inhibition and ATPase stimulation.

Dacomitinib antagonizes multidrug resistance (MDR) in cancer cells by inhibiting the efflux activity of ABCB1 and ABCG2 transporters

The development of multidrug resistance (MDR) to chemotherapy remains a major challenge in the treatment of cancer. Numerous mechanisms have been recognized that cause MDR, but one of the most important mechanisms is overexpression of adenosine triphosphate (ATP)-binding cassette (ABC) transporters, through which the efflux of various anticancer drugs against their concentration gradients is powered by ATP. In recent years, small molecular tyrosine kinase inhibitors (TKIs) have been developed for treatment in various human cancers overexpressing epidermal growth factor receptor (EGFR). At the same time, some TKIs have been shown to be capable of inhibiting ABC transporter-mediated MDR. Dacomitinib (PF-00299804) is a second generation, irreversible TKI, which has shown positive anticancer activities in some preclinical and clinical trials. As many TKIs are substrates or inhibitors of ABC transporters, this study investigates whether dacomitinib could interact with ABC subfamily members that mediate MDR, including ABCB1 (P-gp), ABCG2 (BCRP) and ABCC1 (MRP1). The results showed that dacomitinib at 1.0 μM significantly reversed drug resistance mediated by ABCB1 and ABCG2, but not ABCC1, doing so by antagonizing the drug efflux function in ABCB1- and ABCG2-overexpressing cell lines. The reversal effect on ABCB1-overexpressing cells is more potent than that on ABCG2-overexpressing cells. In addition, dacomitinib at reversal concentration affected neither the protein expression level nor the localization of ABCB1 and ABCG2. Therefore, the mechanisms of this modulating effect are likely to be the following: first, as an inhibitor of ABCB1 or ABCG2 transporters, dacomitinib binds to drug-substrate site in transmembrane domains (TMD) stably in a noncompetitive manner; or second, dacomitinib inhibits ATPase activity and maintains the stability of TMD conformation in a concentration-dependent manner thereby inhibiting the drug efflux function of ABCB1 or ABCG2 transporter. This study provides a useful combinational therapeutic strategy with dacomitinib and substrates of ABCB1 and/or ABCG2 transporters in ABCB1- or ABCG2-overexpressing cancers.

Abstract 4063: Design and synthesis of indole derivatives as ABCG2 mediated resistance reversal agents

Abstract Multidrug resistance is a phenomenon that cancer cells become resistant to anticancer drugs. Multidrug resistance has been shown to be related with the overexpression of ATP-binding (ABC) transporters, including ABCG2. Therefore, ABCG2 is a therapeutic target for the development of multidrug resistance reversal agents. Fumitremorgin C, with an indole group which is an important pharmacophore in many drugs, is a known ABCG2 inhibitor. Inspired by this, we synthesized two series of indole derivatives as ABCG2 mediated resistance reversal agents. With MTT assay, we found that two compounds from the two series of indole derivatives respectively exhibited good reversal activity against ABCG2. The two compounds did not significantly alter the expression of ABCG2 in ABCG2-overexpressing cell lines and they stimulated the basal ATP hydrolysis of ABCG2. Citation Format: Chao-Yun Cai, Hong Zhai, Zi-Ning Lei, Bao-Li Chen, Zhao-Yi Du, Yun-Kai Zhang, Yi-Jun Wang, Bo Wang, Zhe-Sheng Chen. Design and synthesis of indole derivatives as ABCG2 mediated resistance reversal agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4063. doi:10.1158/1538-7445.AM2017-4063

Unexpected Lower Expression of Oncoprotein Gankyrin in Drug Resistant ABCG2 Overexpressing Breast Cancer Cell Lines

Background:Development of a multidrug resistance (MDR) phenotype to chemotherapy remains a major barrier in the treatment of cancer. Gankyrin(p28, p28GANKor PSMD10) is an oncoprotein overexpressed in different carcinoma cell lines. The aim of this study was to compare Gankyrin expression level in MDR cells (MCF-7/ADR and MCF-7/MX) and non-MDR counterparts (MCF-7).Methods:Gankyrin, MDR1 (also known as ABCB1; the ATP-binding cassette sub-family B member 1) and ABCG2(also known as BCRP; the human breast cancer resistance protein) mRNA levels were analyzed by real-time RT-PCR. Western blot analysis was used to detect the protein expression levels of Gankyrin.Results:The PCR results showed that the expression of Gankyrin was significantly lower in the ABCG2overexpressing cell line MCF-7/MX than in non-resistanct MCF-7 cells. In contrast, there were no significant differences in mRNA expression of Gankyrin in the MDR1 overexpressing cell line MCF-7/ADR in comparison with MCF-7 cells. Similarly, Western blot analysis confirmed lower expression of Gankyrin protein in the MCF-7/MX cell line (26% compared to controls) but not in MCF-7/ADR cells.Conclusion:These findings showed that there may be a relation between down-regulation of Gankyrin and overexpression of ABCG2 but without any clear relationship with MDR1 expression in breast cancer cell lines.

Abstract 829: The novel camptothecin derivative and IAP inhibitor FL118 is an effective treatment for irinotecan-refractory colorectal cancer

Abstract The current standard care for metastatic colorectal cancer is combination of leucovorin, 5-fluorouracil, and irinotecan. Irinotecan is a semisynthetic analogue of the alkaloid camptothecin and functions by inducing DNA double-strand breaks through inhibition of topoisomerase I (Top1) activity. The camptothecin-derived Top1 inhibitors irinotecan and topotecan are effective anti-cancer agents that are currently utilized as first-line therapies in several human cancers. Unfortunately, resistance to these agents usually develops during treatment, often through upregulation of the drug efflux pump ABCG2, an ATP-binding cassette transporter. By increasing the rate of drug export, ABCG2 decreases the amount of irinotecan or topotecan that accumulates intracellularly, thereby protecting cancer cells from the cytotoxic effects of these drugs. Therefore, a camptothecin derivative that is also a poor substrate for ABCG2 could potentially overcome ABCG2-induced irinotecan resistance and restore therapeutic efficacy. Our lab recently reported on a novel camptothecin derivative, FL118, which shows strong anti-cancer activity both in vitro and in vivo. Here we report the ability of FL118 to overcome irinotecan resistance. Several irinotecan-resistant colon cancer cell lines derived from HCT116 were treated with either FL118 or SN38, the active metabolite of irinotecan. We observed a decrease in the potency of SN38 in irinotecan-resistant cells that overexpressed ABCG2 compared to cells that did not overexpress ABCG2. In contrast, this loss of potency was not observed for FL118. To confirm that the decrease in potency was ABCG2-dependent, HCT116-A2, an ABCG2 overexpressing cell line, was treated with SN38 or FL118 in the presence or absence of Ko143, an ABCG2 inhibitor. We observed that Ko143 could restore potency to SN38 in HCT116-A2 cells, confirming that irinotecan resistance in HCT116-A2 cells is mediated by ABCG2. However, Ko143 could not modulate the potency of FL118, further suggesting that FL118 is not affected by ABCG2 activity. Similar results were observed in studies with anti-ABCG2 shRNA. Based on these observations, we conclude that FL118 is not a substrate for ABCG2, and that further testing is warranted to determine whether FL118 would be an effective anti-cancer agent for patients with irinotecan-resistant colorectal cancer overexpressing ABCG2. Citation Format: David Westover, Xiang Ling, Xiaojun Liu, Hong Lam, Celine Gongora, Maguy Del Rio, Fengzhi Li. The novel camptothecin derivative and IAP inhibitor FL118 is an effective treatment for irinotecan-refractory colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 829. doi:10.1158/1538-7445.AM2014-829

ARRY-334543 reverses multidrug resistance by antagonizing the activity of ATP-binding cassette subfamily G member 2.

ARRY-334543 is a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases. We conducted this study to determine whether ARRY-334543 can enhance the efficacy of conventional anticancer drugs through interaction with ABC transporters. Lung cancer cell line NCI-H460 and its ABCG2-overexpressing NCI-H460/MX20, as well as the ABCG2-, ABCB1-, and ABCC10-overexpressing transfected cell lines were used for the reversal study. Our results demonstrated that ARRY-334543 (1.0 μM) significantly reversed ABCG2-mediated multidrug resistance (MDR) by directly inhibiting the drug efflux function of ABCG2, resulting in the elevated intracellular accumulation of chemotherapeutic drugs in the ABCG2-overexpressing cell lines. In addition, in isolated membranes, ARRY-334543 stimulated ATPase activity and inhibited photolabeling of ABCG2 with [(125)I]-iodoarylazidoprazosin in a concentration-dependent manner indicating that this drug directly interacts at the drug-binding pocket of this transporter. ARRY-334543 (1.0 μM) only slightly reversed ABCB1- and partially reversed ABCC10-mediated MDR suggesting that it exhibits high affinity toward ABCG2. Moreover, homology modeling predicted the binding conformation of ARRY-334543 at Arg482 centroid-based grid of ABCG2. However, ARRY-334543 at reversal concentrations did not affect the expression level of ABCG2, AKT and ERK1/2 and regulate the re-localization of ABCG2. We conclude that ARRY-334543 significantly reverses drug resistance mediated by ABCG2.

Icotinib antagonizes ABCG2-mediated multidrug resistance, but not the pemetrexed resistance mediated by thymidylate synthase and ABCG2.

ABCG2 is a potential biomarker causing multidrug resistance (MDR) in Non-Small Cell Lung Cancer (NSCLC). We conducted this study to investigate whether Icotinib, a small-molecule inhibitor of EGFR tyrosine kinase, could interact with ABCG2 transporter in NSCLC. Our results showed that Icotinib reversed ABCG2-mediated MDR by antagonizing the drug efflux function of ABCG2. Icotinib stimulated the ATPase activity in a concentration-dependent manner and inhibited the photolabeling of ABCG2 with [125I]-Iodoarylazidoprazosin, demonstrating that it interacts at the drug-binding pocket. Homology modeling predicted the binding conformation of Icotinib at Asn629 centroid-based grid of ABCG2. However, Icotinib at reversal concentration did not affect the expression levels of AKT and ABCG2. Furthermore, a combination of Icotinib and topotecan exhibited significant synergistic anticancer activity against NCI-H460/MX20 tumor xenografts. However, the inhibition of transport activity of ABCG2 was insufficient to overcome pemetrexed resistance in NCI-H460/MX20 cells, which was due to the co-upregulated thymidylate synthase (TS) and ABCG2 expression. This is the first report to show that the up-regulation of TS in ABCG2-overexpressing cell line NCI-H460/MX20 may play a role of resistance to pemetrexate. Our findings suggested different possible strategies of overcoming the resistance of topotecan and pemetrexed in the NSCLC patients.

Telatinib reverses chemotherapeutic multidrug resistance mediated by ABCG2 efflux transporter in vitro and in vivo

Multidrug resistance (MDR) is a phenomenon where cancer cells become simultaneously resistant to anticancer drugs with different structures and mechanisms of action. MDR has been shown to be associated with overexpression of ATP-binding cassette (ABC) transporters. Here, we report that telatinib, a small molecule tyrosine kinase inhibitor, enhances the anticancer activity of ABCG2 substrate anticancer drugs by inhibiting ABCG2 efflux transporter activity. Co-incubation of ABCG2-overexpressing drug resistant cell lines with telatinib and ABCG2 substrate anticancer drugs significantly reduced cellular viability, whereas telatinib alone did not significantly affect drug sensitive and drug resistant cell lines. Telatinib at 1μM did not significantly alter the expression of ABCG2 in ABCG2-overexpressing cell lines. Telatinib at 1μM significantly enhanced the intracellular accumulation of [3H]-mitoxantrone (MX) in ABCG2-overexpressing cell lines. In addition, telatinib at 1μM significantly reduced the rate of [3H]-MX efflux from ABCG2-overexpressing cells. Furthermore, telatinib significantly inhibited ABCG2-mediated transport of [3H]-E217βG in ABCG2 overexpressing membrane vesicles. Telatinib stimulated the ATPase activity of ABCG2 in a concentration-dependent manner, indicating that telatinib might be a substrate of ABCG2. Binding interactions of telatinib were found to be in transmembrane region of homology modeled human ABCG2. In addition, telatinib (15mg/kg) with doxorubicin (1.8mg/kg) significantly decreased the growth rate and tumor size of ABCG2 overexpressing tumors in a xenograft nude mouse model. These results, provided that they can be translated to humans, suggesting that telatinib, in combination with specific ABCG2 substrate drugs may be useful in treating tumors that overexpress ABCG2.

Tivozanib reverses multidrug resistance mediated by ABCB1 (P-glycoprotein) and ABCG2 (BCRP)

This study aimed to investigate the mechanism of reversal of multidrug resistance mediated by ABC transporters with tivozanib (AV-951 and KRN-951). Tivozanib is a potent inhibitor of VEGF-1, -2 and -3 receptors. ABCB1- and ABCG2-overexpressing cell lines were treated with respective substrate antineoplastic agents in the presence or absence of tivozanib. The results indicate that tivozanib can significantly reverse ABCB1-mediated resistance to paclitaxel, vinblastine and colchicine, as well as ABCG2-mediated resistance to mitoxantrone, SN-38 and doxorubicin. Drug efflux assays showed that tivozanib increased the intracellular accumulation of substrates by inhibiting the ABCB1 and ABCG2 efflux activity. Furthermore, at a higher concentration, tivozanib inhibited the ATPase activity of both ABCB1 and ABCG2 and inhibited the photolabeling of ABCB1 or ABCG2. We conclude that tivozanib at noncytotoxic concentrations has the previously unknown activity of reversing multidrug resistance mediated by ABCB1 and ABCG2 transporters.

Abstract 1443: Pharmacokinetics and pharmacogenetics of Actinomycin D in an animal model and pediatric patient population

Abstract ATP binding cassette (ABC) transporters, such as ABCB1 (MDR1/P-gp), are expressed throughout the body to protect against exogenous toxins. ABC transporters control removal from the body of anti-cancer drugs such as doxorubicin and etoposide via biliary or renal excretion. Actinomycin D (Act D) has been used successfully to treat adult and childhood cancers for over 30 years. It continues to play a key role in the treatment of Wilms tumour patients, with 5-year survival rates >80% currently observed. Although Act D is generally well tolerated, severe hepatic toxicity has been associated with treatment. It has previously been demonstrated that Act D exposure is associated with a large degree of inter-patient variability in children with cancer. As common single nucleotide polymorphisms (SNPs) in ABC transporters have previously been shown to alter clinical exposure to various anti-cancer agents, we have carried out translational and clinical studies to investigate the transport of Act D using well established cell line and animal models. Growth inhibition (GI) and intracellular accumulation studies, over a range of Act D concentrations (0-10µM), were initially carried out to determine the role of ABC transporter expression in MDCKII-WT and ABCB1, ABCC1, ABCC2 and ABCG2 over-expressing cell lines. To assess the importance of ABCB1 and ABCC2 transporter expression in vivo, Act D pharmacokinetics and exposure were determined in Abcb1a/1b and Abcc2 knockout mice over 6 hours. In addition, liver, kidney and brain tissue samples were analysed to assess Act D tissue accumulation. Further pharmacokinetic sampling has also been carried out from children receiving Act D as part of their standard treatment, with analysis using a validated LC/MS method. Blood samples were obtained from all patients for pharmacogenetic analysis. Following treatment with Act D, a 59-fold decrease in Act D sensitivity was observed in ABCB1 over-expressing cells (GI50: 745nM), a 2-fold decrease in ABCC1 (GI50: 25.7nM) and a 3-fold decrease (GI50: 40.4nM) in ABCC2, as compared to the parental cell line (GI50: 12.7nM). Intracellular accumulation of Act D was lower in ABCB1 cells compared to the parental cell line, following incubation with Act D for 6 hours. This effect was abrogated by use of the ABCB1 inhibitor verapamil. In vivo pharmacokinetic analysis revealed a 64% higher AUC0-6h in mice with no Abcb1a/1b expression and a 22% lower AUC0-6h in Abcc2-/- mice compared to WT. Pharmacokinetic analysis of samples obtained from 71 patients indicated a large inter-patient variability in Act D exposure (AUC0-24h range: 1.7 - 19.4 µg/ml.min). Ongoing investigations into pharmacogenetic variation in ABCB1 and ABCC2 tagSNPs in these patients provide novel preliminary data relating to links between ABCB1 haplotypes and Act D exposure in children with cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1443. doi:1538-7445.AM2012-1443

Abstract 5614: Tandutinib (MLN518/CT53518) targeted to stem-like cells by inhibiting the function of ATP-binding cassette subfamily G member 2

Abstract Tandutinib (MLN518/CT53518) is a novel quinazoline-based inhibitor of FMS-like tyrosine kinase 3, platelet-derived growth factor receptor, and KIT. In this study, we evaluated the possible interactions of tandutinib with P-glycoprotein (P-gp/ABCB1), multidrug resistance protein 1 (MRP1/ABCC1), breast cancer resistance protein (BCRP/ABCG2) and MRP4/ABCC4. Our results showed that tandutinib reverses ABCG2-mediated drug resistance in ABCG2-transfected cell lines ABCG2-482-R2, ABCG2-482-G2 and ABCG2-482-T7 and mitoxantrone-induced ABCG2-overexpressing cell line S1-M1-80. On the other hand, tandutinib did not change the sensitivity of all tested ABCG2-overexpressing cell lines and their sensitive parental cell lines to cisplatin (non-substrate of ABCG2) and had no significant reversal effect on ABCB1-, ABCC1- and ABCC4-mediated drug resistance. Tandutinib increased doxorubicin and rhodamine 123 accumulation in S1-M1-80 cells in a dose-dependent manner, but did not change their accumulation in parental S1 cells. Similarly, tandutinib also enhanced [3H]mitoxantone accumulation in ABCG2-transfected cell lines, significantly inhibited mitoxantrone efflux in ABCG2-482-R2 cells, but not in HEK293/pcDNA 3.1 cells. However, tandutinib did not stimulate the ATPase activity of ABCG2 and significantly inhibit the photolabeling of ABCG2 with [125I]Iodoarylazidoprazosin. Tandutinib also did not affect the expression of ABCG2 at mRNA or protein levels. In addition, tandutinib did not block the phosphorylation of Akt and Erk1/2 in S1-M1-80 or S1 cells. Importantly, tandutinib targeted to A549 side population cells and enhanced their sensitivity to mitoxantrone, which was not altered in non-SP cells. Collectively, our results advocate the use of tandutinib as an ABCG2 modulator and stem cell targeted agent to increase efficiency of anticancer drugs in clinic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5614. doi:1538-7445.AM2012-5614

Dasatinib Cellular Uptake and Efflux in Chronic Myeloid Leukemia Cells: Therapeutic Implications

The organic cation transporter OCT-1 mediates active transport of imatinib. We recently showed that low OCT-1 activity is a major contributor to suboptimal response in chronic myeloid leukemia (CML) patients treated with imatinib. The relevance of OCT-1 activity and efflux pumps in determining intracellular uptake and retention (IUR) of dasatinib was assessed. The effect of OCT inhibitors on [14C]dasatinib and [14C]imatinib IUR was compared using peripheral blood mononuclear cells from newly diagnosed CML patients. The role of efflux transporters was studied using ABCB1- and ABCG2-overexpressing cell lines and relevant inhibitors. Unlike imatinib, there was no significant difference in the dasatinib IUR at 37 degrees C and 4 degrees C (P = 0.8), and OCT-1 inhibitors including prazosin did not reduce dasatinib IUR significantly. In CML mononuclear cells, prazosin inhibitable IUR was significantly higher for imatinib than dasatinib (6.38 versus 1.48 ng/200,000 cells; P = 0.002; n = 11). Patients with high OCT-1 activity based on their imatinib uptake had IC50(dasatinib) values equivalent to patients with low OCT-1 activity. Dasatinib IUR was significantly lower in ABCB1-overexpressing cell lines compared with parental cell lines (P < 0.05). PSC833 (ABCB1 inhibitor) significantly increased the dasatinib IUR (P < 0.05) and reduced IC50(dasatinib) (from 100 to 8 nmol/L) in K562-DOX cell line. The ABCG2 inhibitor Ko143 significantly increased dasatinib IUR in ABCG2-overexpressing cell lines and reduced IC(50)(dasatinib). Unlike imatinib, dasatinib cellular uptake is not significantly affected by OCT-1 activity, so that expression and function of OCT-1 is unlikely to affect response to dasatinib. Dasatinib is a substrate of both efflux proteins, ABCB1 and ABCG2.

Mutations at amino-acid 482 in the ABCG2 gene affect substrate and antagonist specificity.

Recent studies have shown that mutations at amino-acid 482 in the ABCG2 gene affect the substrate specificity of the protein. To delineate the effects of these mutations clearly, human embryonic kidney cells (HEK-293) were stably transfected with wild-type 482R or mutant 482G and 482T ABCG2. By flow cytometry, mitoxantrone, BODIPY-prazosin, and Hoechst 33342 were found to be substrates of all ABCG2 proteins, while rhodamine 123, daunorubicin, and LysoTracker Green were transported only by mutant ABCG2. In cytotoxicity assays, all ABCG2 proteins conferred high levels of resistance to mitoxantrone, SN-38, and topotecan, while mutant ABCG2 also exhibited a gain of function for mitoxantrone as they conferred a four-fold greater resistance compared to wild type. Cells transfected with mutant ABCG2 were 13- to 71- fold resistant to the P-glycoprotein substrates doxorubicin, daunorubicin, epirubicin, bisantrene, and rhodamine 123 compared to cells transfected with wild-type ABCG2, which were only three- to four-fold resistant to these compounds. ABCG2 did not confer appreciable resistance to etoposide, taxol or the histone deacetylase inhibitor depsipeptide. None of the transfected cell lines demonstrated resistance to flavopiridol despite our previous observation that ABCG2-overexpressing cell lines are cross-resistant to the drug. Recently reported inhibitors of ABCG2 were evaluated and 50 μM novobiocin was found to reverse wild-type ABCG2 completely, but only reverse mutant ABCG2 partially. The studies presented here serve to underscore the importance of amino-acid 482 in defining the substrate specificity of the ABCG2 protein and raise the possibility that amino-acid 482 mutations in human cancers could affect the clinical application of antagonists for ABCG2.