Karthika Natarajan

The Novel BCR-ABL and FLT3 Inhibitor Ponatinib Is a Potent Inhibitor of the MDR-Associated ATP-Binding Cassette Transporter ABCG2

Ponatinib is a novel tyrosine kinase inhibitor with potent activity against BCR-ABL with mutations, including T315I, and also against fms-like tyrosine kinase 3. We tested interactions between ponatinib at pharmacologically relevant concentrations of 50 to 200 nmol/L and the MDR-associated ATP-binding cassette (ABC) proteins ABCB1, ABCC1, and ABCG2. Ponatinib enhanced uptake of substrates of ABCG2 and ABCB1, but not ABCC1, in cells overexpressing these proteins, with a greater effect on ABCG2 than on ABCB1. Ponatinib potently inhibited [125I]-IAAP binding to ABCG2 and ABCB1, indicating binding to their drug substrate sites, with IC50 values of 0.04 and 0.63 μmol/L, respectively. Ponatinib stimulated ABCG2 ATPase activity in a concentration-dependent manner and stimulated ABCB1 ATPase activity at low concentrations, consistent with it being a substrate of both proteins at pharmacologically relevant concentrations. The ponatinib IC50 values of BCR-ABL–expressing K562 cells transfected with ABCB1 and ABCG2 were approximately the same as and 2-fold higher than that of K562, respectively, consistent with ponatinib being a substrate of both proteins, but inhibiting its own transport, and resistance was also attenuated to a small degree by ponatinib-induced downregulation of ABCB1 and ABCG2 cell-surface expression on resistant K562 cells. Ponatinib at pharmacologically relevant concentrations produced synergistic cytotoxicity with ABCB1 and ABCG2 substrate chemotherapy drugs and enhanced apoptosis induced by these drugs, including daunorubicin, mitoxantrone, topotecan, and flavopiridol, in cells overexpressing these transport proteins. Combinations of ponatinib and chemotherapy drugs warrant further testing. Mol Cancer Ther; 11(9); 2033–44. ©2012 AACR.

Pim-1 Kinase Phosphorylates and Stabilizes 130 kDa FLT3 and Promotes Aberrant STAT5 Signaling in Acute Myeloid Leukemia with FLT3 Internal Tandem Duplication

The type III receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) is expressed on both normal hematopoietic stem cells and acute myeloid leukemia (AML) cells and regulates their proliferation. Internal tandem duplication (ITD) mutation of FLT3 is present in a third of AML cases, results in constitutive activation and aberrant signaling of FLT3, and is associated with adverse treatment outcomes. While wild-type (WT) FLT3 is predominantly a 150 kDa complex glycosylated cell surface protein, FLT3-ITD is partially retained in the endoplasmic reticulum as a 130 kDa underglycosylated species associated with the chaperones calnexin and heat shock protein (HSP) 90, and mediates aberrant STAT5 signaling, which upregulates the oncogenic serine/threonine kinase Pim-1. FLT3 contains a Pim-1 substrate consensus serine phosphorylation site, and we hypothesized that it might be a Pim-1 substrate. Pim-1 was indeed found to directly interact with and serine-phosphorylate FLT3. Pim-1 inhibition decreased the expression and half-life of 130 kDa FLT3, with partial abrogation by proteasome inhibition, in association with decreased FLT3 binding to calnexin and HSP90, and increased 150 kDa FLT3 expression and half-life, with abrogation by inhibition of glycosylation. These findings were consistent with Pim-1 stabilizing FLT3-ITD as a 130 kDa species associated with calnexin and HSP90 and inhibiting its glycosylation to form the 150 kDa species. Pim-1 knockdown effects were similar. Pim-1 inhibition also decreased phosphorylation of FLT3 at tyrosine 591 and of STAT5, and expression of Pim-1 itself, consistent with inhibition of the FLT3-ITD-STAT5 signaling pathway. Finally, Pim-1 inhibition synergized with FLT3 inhibition in inducing apoptosis of FLT3-ITD cells. This is, to our knowledge, the first demonstration of a role of Pim-1 in a positive feedback loop promoting aberrant signaling in malignant cells.

The FLT3 Inhibitor Quizartinib Inhibits ABCG2 at Pharmacologically Relevant Concentrations, with Implications for Both Chemosensitization and Adverse Drug Interactions

The oral second-generation bis-aryl urea fms-like tyrosine kinase 3 (FLT3) inhibitor quizartinib (AC220) has favorable kinase selectivity and pharmacokinetics. It inhibits mutant and wild-type FLT3 in vivo at 0.1 and 0.5 µM, respectively, and has shown favorable activity and tolerability in phase I and II trials in acute myeloid leukemia, with QT prolongation as the dose-limiting toxicity. Co-administration with chemotherapy is planned. We characterized interactions of quizartinib with the ATP-binding cassette (ABC) proteins ABCB1 (P-glycoprotein) and ABCG2 (breast cancer resistance protein). Its effects on uptake of fluorescent substrates and apoptosis were measured by flow cytometry, binding to ABCB1 and ABCG2 drug-binding sites by effects on [125I]iodoarylazidoprazosin ([125I]-IAAP) photolabeling and ATPase activity, and cell viability by the WST-1 colorimetric assay. Quizartinib inhibited transport of fluorescent ABCG2 and ABCB1 substrates in ABCG2- and ABCB1-overexpressing cells in a concentration-dependent manner, from 0.1 to 5 µM and from 0.5 to 10 µM, respectively, and inhibited [125I]-IAAP photolabeling of ABCG2 and ABCB1 with IC50 values of 0.07 and 3.3 µM, respectively. Quizartinib at higher concentrations decreased ABCG2, but not ABCB1, ATPase activity. Co-incubation with quizartinib at 0.1 to 1 µM sensitized ABCG2-overexpressing K562/ABCG2 and 8226/MR20 cells to ABCG2 substrate chemotherapy drugs in a concentration-dependent manner in cell viability and apoptosis assays. Additionally, quizartinib increased cellular uptake of the ABCG2 substrate fluoroquinolone antibiotic ciprofloxacin, which also prolongs the QT interval, in a concentration-dependent manner, predicting altered ciprofloxacin pharmacokinetics and pharmacodynamics when co-administered with quizartinib. Thus quizartinib inhibits ABCG2 at pharmacologically relevant concentrations, with implications for both chemosensitization and adverse drug interactions. These interactions should be considered in the design of treatment regimens combining quizartinib and chemotherapy drugs and in choice of concomitant medications to be administered with quizartinib.

Hydroxylated Dimeric Naphthoquinones Increase the Generation of Reactive Oxygen Species, Induce Apoptosis of Acute Myeloid Leukemia Cells and Are Not Substrates of the Multidrug Resistance Proteins ABCB1 and ABCG2.

Selective targeting of the oxidative state, which is a tightly balanced fundamental cellular property, is an attractive strategy for developing novel anti-leukemic chemotherapeutics with potential applications in the treatment of acute myeloid leukemia (AML), a molecularly heterogeneous disease. Dimeric naphthoquinones (BiQs) with the ability to undergo redox cycling and to generate reactive oxygen species (ROS) in cancer cells are a novel class of compounds with unique characteristics that make them excellent candidates to be tested against AML cells. We evaluated the effect of two BiQ analogues and one monomeric naphthoquinone in AML cell lines and primary cells from patients. All compounds possess one halogen and one hydroxyl group on the quinone cores. Dimeric, but not monomeric, naphthoquinones demonstrated significant anti-AML activity in the cell lines and primary cells from patients with favorable therapeutic index compared to normal hematopoietic cells. BiQ-1 effectively inhibited clonogenicity and induced apoptosis as measured by Western blotting and Annexin V staining and mitochondrial membrane depolarization by flow cytometry. BiQ-1 significantly enhances intracellular ROS levels in AML cells and upregulates expression of key anti-oxidant protein, Nrf2. Notably, systemic exposure to BiQ-1 was well tolerated in mice. In conclusion, we propose that BiQ-induced therapeutic augmentation of ROS in AML cells with dysregulation of antioxidants kill leukemic cells while normal cells remain relatively intact. Further studies are warranted to better understand this class of potential chemotherapeutics.

Functional cyclic AMP response element in the breast cancer resistance protein (BCRP/ABCG2) promoter modulates epidermal growth factor receptor pathway- or androgen withdrawal-mediated BCRP/ABCG2 transcription in human cancer cells

Phosphorylated cyclic-AMP (cAMP) response element binding protein (p-CREB) is a downstream effector of a variety of important signaling pathways. We investigated whether the human BCRP promoter contains a functional cAMP response element (CRE). 8Br-cAMP, a cAMP analogue, increased the activity of a BCRP promoter reporter construct and BCRP mRNA in human carcinoma cells. Epidermal growth factor receptor (EGFR) pathway activation also led to an increase in p-CREB and in BCRP promoter reporter activity via two major downstream EGFR signaling pathways: the phosphotidylinositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase (MAPK) pathway. EGF treatment increased the phosphorylation of EGFR, AKT, ERK and CREB, while simultaneously enhancing BCRP mRNA and functional protein expression. EGF-stimulated CREB phosphorylation and BCRP induction were diminished by inhibition of EGFR, PI3K/AKT or RAS/MAPK signaling. CREB silencing using RNA interference reduced basal levels of BCRP mRNA and diminished the induction of BCRP by EGF. Chromatin immunoprecipitation assays confirmed that a putative CRE site on the BCRP promoter bound p-CREB by a point mutation of the CRE site abolished EGF-induced stimulation of BCRP promoter reporter activity. Furthermore, the CREB co-activator, cAMP-regulated transcriptional co-activator (CRTC2), is involved in CREB-mediated BCRP transcription: androgen depletion of LNCaP human prostate cancer cells increased both CREB phosphorylation and CRTC2 nuclear translocation, and enhanced BCRP expression. Silencing CREB or CRTC2 reduced basal BCRP expression and BCRP induction under androgen-depletion conditions. This novel CRE site plays a central role in mediating BCRP gene expression in several human cancer cell lines following activation of multiple cancer-relevant signaling pathways.

Pim kinase inhibition sensitizes FLT3-ITD acute myeloid leukemia cells to topoisomerase 2 inhibitors through increased DNA damage and oxidative stress

Internal tandem duplication of fms-like tyrosine kinase-3 (FLT3-ITD) is frequent (30 percent) in acute myeloid leukemia (AML), and is associated with short disease-free survival following chemotherapy. The serine threonine kinase Pim-1 is a pro-survival oncogene transcriptionally upregulated by FLT3-ITD that also promotes its signaling in a positive feedback loop. Thus inhibiting Pim-1 represents an attractive approach in targeting FLT3-ITD cells. Indeed, co-treatment with the pan-Pim kinase inhibitor AZD1208 or expression of a kinase-dead Pim-1 mutant sensitized FLT3-ITD cell lines to apoptosis triggered by chemotherapy drugs including the topoisomerase 2 inhibitors daunorubicin, etoposide and mitoxantrone, but not the nucleoside analog cytarabine. AZD1208 sensitized primary AML cells with FLT3-ITD to topoisomerase 2 inhibitors, but did not sensitize AML cells with wild-type FLT3 or remission bone marrow cells, supporting a favorable therapeutic index. Mechanistically, the enhanced apoptosis observed with AZD1208 and topoisomerase 2 inhibitor combination treatment was associated with increased DNA double-strand breaks and increased levels of reactive oxygen species (ROS), and co-treatment with the ROS scavenger N-acetyl cysteine rescued FLT3-ITD cells from AZD1208 sensitization to topoisomerase 2 inhibitors. Our data support testing of Pim kinase inhibitors with topoisomerase 2 inhibitors, but not with cytarabine, to improve treatment outcomes in AML with FLT3-ITD.

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1.

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique - usually untranslated - first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

Role of Breast Cancer Resistance Protein (BCRP, ABCG2) in Cancer Outcomes and Drug Resistance

The breast cancer resistance protein (BCRP), formally known as ATP-binding cassette protein G2 (ABCG2), is an efflux transporter that plays a significant role in altering absorption, distribution, metabolism, and excretion (ADME) of most extant and emerging molecular cancer therapeutics. BCRP expressed by neoplastic cells may also contribute to the resistance of these cells to chemotherapeutic agents. Although the expression of BCRP in human cancers has often correlated with adverse outcomes, to date therapeutic strategies utilizing the inhibition of BCRP function to improve the ADME of cancer chemotherapeutics or to sensitize cancer cells that express BCRP to chemotherapy have not been fruitful. This review will examine the most current literature probing BCRP’s role in ADME of cancer therapeutic agents and drug resistance.

Abstract 4581: Pim-1 kinase inhibition in the absence of FLT3 inhibition results in cytostatic effects in acute myeloid leukemia cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The serine threonine kinase Pim-1 regulates expression of proteins involved in cell cycle, proliferation, apoptosis and drug resistance. Pim-1 has been identified as a potential therapeutic target in acute myeloid leukemia (AML). Notably, it is overexpressed downstream of FLT3 with internal tandem duplication (ITD), present in 30% of AML cases and associated with adverse treatment outcomes. SGI-1776 is the only Pim inhibitor in clinical testing to date, but testing was discontinued due to QTc prolongation. SGI-1776 has been used in laboratory studies of AML, but is also a FLT3 inhibitor, and effects of Pim-1 and FLT3 inhibition have been difficult to distinguish, particularly in cells with FLT3-ITD. To better characterize the effects of Pim-1 inhibition in AML, we compared SGI-1776 (from SuperGen, Inc., Dublin, CA) with two Pim inhibitors without FLT3 inhibitory activity, SMI-4a and quercetagetin in FLT3-wt and FLT3-ITD cells in 96-hour WST-1 cell viability, flow cytometric cell cycle and apoptosis, colony formation and liquid culture proliferation assays. SGI-1776 had no effect on cell cycle in FLT3-wt cells at concentrations up to 5 μM, and caused apoptosis at 10 μM, while G1 arrest and apoptosis occurred at 100 nM in FLT3-ITD cells, and SMI-4a and quercetagetin did not alter cell cycle nor induce apoptosis in FLT3-wt nor FLT3-ITD cell lines. IC50 (μM) comparisons are summarized below: In liquid culture assays, all three Pim inhibitors slowed proliferation of all cell lines, indicating a cytostatic effect. Thus FLT3 inhibition plays a substantial role in the cellular effects of SGI-1776, and Pim inhibitors without FLT3 inhibitory activity appear to have primarily cytostatic effects in AML cells. Other Pim kinase inhibitors in development should be characterized with regard to pro-apoptotic versus cytostatic effects. ![Figure][1] 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 4581. doi:1538-7445.AM2012-4581 [1]: pending:yes

Abstract 706: The serine/threonine kinase Pim-1 promotes drug resistance mediated by the ATP-binding cassette multidrug resistance protein breast cancer resistance protein (BCRP, ABCG2) by stabilizing higher-order BCRP multimers

Background: The ATP-binding cassette (ABC) multidrug resistance protein breast cancer resistance protein (BCRP, ABCG2) is a half-transporter that was thought to function as a homodimer, but has been shown to exist as a homotetramer in the membranes of drug resistant cells and to also form higher-order multimers. The serine/threonine kinase Pim-1, expressed in both hematologic malignancies and solid tumors, phosphorylates BCRP at T362 and promotes its multimerization and drug efflux function. Inhibiting Pim-1 sensitizes BCRP-overexpressing cells to BCRP substrate chemotherapy drugs. Pim-1 phosphorylation has been previously shown to stabilize some of its substrate proteins, but the mechanism of its effect on BCRP is unknown. We sought to delineate the mechanism of Pim-1 effect on BCRP. Methods: BCRP-overexpressing 8226/MR20 myeloma cells were treated with the protein translation inhibitor cycloheximide (10µg/ml) in the presence and absence of the Pim-1 inhibitor SGI-1776 (SuperGen, Inc., Dublin, CA) (1μM) for 0, 2, 4, 6 and 8 hours, and BCRP levels were measured by densitometric analysis, normalized for GAPDH levels, following Native PAGE as well as SDS-PAGE electrophoresis and immunoblotting. 8226/MR20 cells were also treated with the proteasome inhibitor MG-132 (2 μM) or the lysosome inhibitor bafilomycin A1 (10 nM) in the presence of cycloheximide and in the presence and absence of SGI-1776. Results: Under Native PAGE electrophoresis, BCRP migrated predominantly as a tetramer, with lower levels of higher-order multimers. Half-lives were approximately 6 and 2 hours, respectively. In the presence of SGI-1776, the half-life of higher-order BCRP multimers decreased to approximately 1 hour, while BCRP tetramer levels increased, consistent with destabilization of higher-order BCRP multimers and conversion to tetramers. Proteasomal inhibition with MG-132 and to a lesser extent lysosomal inhibition with bafilomycin A1 increased the half-life of both BCRP tetramers and higher order multimers. However, on co-incubation with SGI-1776, bafilomycin A1 caused marked stabilization of higher-order BCRP multimers, with a lesser effect on tetramers, while proteasomal inhibition with MG-132 had no effect, consistent with Pim-1 protecting high-order multimers from lysosomal degradation. Finally, BCRP dimers measured by SDS-PAGE electrophoresis under non-reducing conditions increased with time during cycloheximide treatment and the increase was more rapid in the presence of SGI-1776, consistent with more rapid destabilization of BCRP multimers when Pim-1 kinase was inhibited. Conclusion: Phosphorylation of BCRP by Pim-1 stabilizes BCRP higher-order multimers, protecting them from dissociation and degradation in the lysosome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 706. doi:10.1158/1538-7445.AM2011-706