Intracellular Uptake And Retention Research Articles

Solute Carrier Family 29A1 Mediates In Vitro Resistance to Azacitidine in Acute Myeloid Leukemia Cell Lines.

Azacitidine (AZA) is commonly used hypomethylating agent for higher risk myelodysplastic syndromes and acute myeloid leukemia (AML). Although some patients achieve remission, eventually most patients fail AZA therapy. Comprehensive analysis of intracellular uptake and retention (IUR) of carbon-labeled AZA (14C-AZA), gene expression, transporter pump activity with or without inhibitors, and cytotoxicity in naïve and resistant cell lines provided insight into the mechanism of AZA resistance. AML cell lines were exposed to increasing concentrations of AZA to create resistant clones. 14C-AZA IUR was significantly lower in MOLM-13- (1.65 ± 0.08 ng vs. 5.79 ± 0.18 ng; p < 0.0001) and SKM-1- (1.10 ± 0.08 vs. 5.08 ± 0.26 ng; p < 0.0001) resistant cells compared to respective parental cells. Importantly, 14C-AZA IUR progressively reduced with downregulation of SLC29A1 expression in MOLM-13- and SKM-1-resistant cells. Furthermore, nitrobenzyl mercaptopurine riboside, an SLC29A inhibitor, reduced 14C-AZA IUR in MOLM-13 (5.79 ± 0.18 vs. 2.07 ± 0.23, p < 0.0001) and SKM-1-naive cells (5.08 ± 2.59 vs. 1.39 ± 0.19, p = 0.0002) and reduced efficacy of AZA. As the expression of cellular efflux pumps such as ABCB1 and ABCG2 did not change in AZA-resistant cells, they are unlikely contribute to AZA resistance. Therefore, the current study provides a causal link between in vitro AZA resistance and downregulation of cellular influx transporter SLC29A1.

Lysosomal Sequestration Determines Intracellular Imatinib Levels.

The intracellular uptake and retention (IUR) of imatinib is reported to be controlled by the influx transporter SLC22A1 (organic cation transporter 1). We recently hypothesized that alternative uptake and/or retention mechanisms exist that determine intracellular imatinib levels. Here, we systematically investigate the nature of these mechanisms. Imatinib uptake in cells was quantitatively determined by liquid chromatography-tandem mass spectrometry. Fluorescent microscopy was used to establish subcellular localization of imatinib. Immunoblotting, cell cycle analyses, and apoptosis assays were performed to evaluate functional consequences of imatinib sequestration. Uptake experiments revealed high intracellular imatinib concentrations in HEK293, the leukemic cell lines K562 and SD-1, and a gastrointestinal stromal tumor cell line GIST-T1. We demonstrated that imatinib IUR is time-, dose-, temperature-, and energy-dependent and provide evidence that SLC22A1 and other potential imatinib transporters do not substantially contribute to the IUR of imatinib. Prazosin, amantadine, NH4Cl, and the vacuolar ATPase inhibitor bafilomycin A1 significantly decreased the IUR of imatinib and likely interfere with lysosomal retention and accumulation of imatinib. Costaining experiments with LysoTracker Red confirmed lysosomal sequestration of imatinib. Inhibition of the lysosomal sequestration had no effect on the inhibition of c-Kit signaling and imatinib-mediated cell cycle arrest but significantly increased apoptosis in imatinib-sensitive GIST-T1 cells. We conclude that intracellular imatinib levels are primarily determined by lysosomal sequestration and do not depend on SLC22A1 expression.

ATP Dependent Efflux Transporters ABCB1 and ABCG2 Are Unlikely to Impact the Efficacy, or Mediate Resistance to the Tyrosine Kinase Inhibitor, Ponatinib

Abstract 2745Ponatinib is a potent pan-BCR-ABL tyrosine kinase inhibitor (TKI) currently in a pivotal phase 2 clinical trial. Ponatinib (PON) was specifically designed to target both native and all mutant forms of BCR-ABL, including T315I. The phase I study of oral ponatinib in patients with refractory CML/ALL or other hematologic malignancies recently reported that 66% and 53% of patients with CP-CML achieved MCyR and CCyR respectively (Cortes et al., ASH 2011 abstract #210).While extensive modelling experiments in BaF3 cells have been performed characterising in vitro response to ponatinib, little is known about the interactions of this drug and drug transporters that impact the response of other tyrosine kinase inhibitors (TKIs). To explore this we have examined both the degree of in vitro kinase inhibition mediated by ponatinib in BCR-ABL+ cell lines, and the intracellular uptake and retention (IUR) of ponatinib achieved. The IC50 was determined by assessing the reduction in %p-Crkl in response to increasing concentrations of ponatinib in vitro. The IUR assay was performed as previously using [14-C]-ponatinib. To determine the role of ABCB1 and ABCG2, both previously implicated in the transport of other TKIs, IC50 analysis was performed on K562 cells, and variants; ABCB1 overexpressing K562-DOX and ABCG2 overexpressing K562-ABCG2. As shown in Table 1, in contrast to the results previously observed with imatinib (IM), nilotinib (NIL) and dasatinib (DAS) there was no significant difference in the IC50ponatinib between these three cell lines, suggesting neither ABCB1 nor ABCG2 play a major role in ponatinib transport. Furthermore, the addition of either the ABCB1 and ABCG2 inhibitor pantoprazole, or the multidrug resistance (MDR) inhibitor cyclosporin did not result in a significant change in the IC50ponatinib in any of the cell lines tested. In contrast the addition of either pantoprazole or cyclosporin resulted in a significant reduction in IC50IM, IC50NIL. and IC50DAS of K562-DOX cells, supporting the notion that these TKIs interact with ABCB1.Table 1:The IC50 of ponatinib (compared to IM, NIL and DAS) in K562 cells and the over-expressing variants DOX and ABCG2 in the presence of the ABC inhibitors pantoprazole and cyclosporin. n=5. *p<0.05IC50% reduction in IC50+ pantoprazole+ cyclosporinPON (nM)IM (μM)NIL (nM)DAS (nM)PONIMNILDASPONIMNILDASK5627.793751111544*NA−107NA2DOX7.919*598*100*1018*63*1655*88*ABCG26.4730025*6NATo further examine the effect of ABC transporters on ponatinib efflux we have determined the IUR of [14-C]-ponatinib in K562, DOX and ABCG2 cell lines. We demonstrate no significant difference in the IUR between these cell lines at 37°C (n=6) (K562 vs DOX p=0.6; K562 vs ABCG2 p=0.37 and DOX vs ABCG2 p=0.667 at 2uM respectively). Temperature dependent IUR experiments reveal a significant reduction in the ponatinib IUR at 4°C compared to 37°C in K562 cells (n=6) (p=0.008), DOX cells (p=0.004) and ABCG2 cells (p=0.002) supporting the likely involvement of an ATP/temperature dependent, and yet to be determined, component of ponatinib influx. There was no significant difference in the IUR between these cell lines at 4°C (p=0.824, p=0.7 and p=0.803 respectively). Importantly, these data are consistent with the IC50ponatinib findings. If ATP dependent efflux pumps (ABCB1 and ABCG2) were actively transporting ponatinib, a significant decrease in IUR in DOX and ABCG2 at 37°C compared to K562 cells would be expected, but is not observed here. Analysis of ponatinib IUR in the prototypic ABCB1 over-expressing CEM-VBL100 cells, and their parental, ABCB1 null counterparts (CCRF-CEM) further confirmed these findings. The IUR in VBL100 cells was significantly higher than that observed in CEM’s (p<0.001; n=5), providing further evidence that ponatinib was not being exported from the cell actively via ABCB1.These data suggest that the transport of ponatinib is, at least in part, temperature-dependent indicating a yet to be determined ATP transporter may be involved in the transport of ponatinib into leukaemic cells. Importantly, this data suggests that ponatinib is unlikely to be susceptible to resistance via the major ATP efflux transporters (ABCB1 or ABCG2) that have been previously demonstrated to significantly impact the transport of, and mediate resistance to other clinically available TKIs. Disclosures:White:BMS: Honoraria, Research Funding; Novartis Pharmaceuticals: Honoraria, Research Funding. Clackson:ARIAD: Employment. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Non-Steroidal Anti-Inflammatory Drugs and Imatinib; Drug Interactions That May Impact Efficacy,

Abstract 3501 Aim:The human organic cation transporter-1 (OCT-1) is the primary active influx protein for imatinib (IM) into target BCR-ABL-positive cells. The functional activity of the OCT-1 protein (OCT-1 activity; OA) is predictive of molecular response and event-free survival in de-novo chronic phase chronic myeloid leukemia (CP-CML) patients. Due to the common use of non-steroidal anti-inflammatory drugs (NSAIDs) among CML patients on IM to treat musculoskeletal pain, it is of particular interest to investigate the impact of NSAIDs on OCT-1 mediated IM uptake. Methods:12 NSAIDs were firstly examined for their impact on OA using the intracellular uptake and retention (IUR) assay and [14-C]-IM. The degree of in vitro kinase inhibition achieved was assessed using p-Crkl in the IC50imatinib assay. Viable cell numbers were determined by trypan blue exclusion method after 72 hours incubation with the co-administration of NSAIDs and IM. NSAIDs concentration was adjusted to median blood levels on standard doses. Results:The majority of NSAIDs (10 of 12) selected did not induce any significant change in OA in CML cell lines. Compared to control cells, increased OA were observed when K562 or KU812 cells were treated with diclofenac (10μM), and this resulted in a concomitant decrease in the IC50imatinib. In keeping with this, after 72 hours co-incubation with IM, diclofenac resulted in a significantly lower viable cell number compared with IM alone. In contrast, ibuprofen (130μM) led to a significant decrease in OA, resulting in an increased IC50imatinib and thus reduced the inhibition of IM on cell growth number (Table 1).Table 1The effect of diclofenac and ibuprofen on the OCT-1 activity, IC50imatinib and viable cell numbers in KU812 and HL60 cellsOCT-1 activity (percentage of vehicle control)IC50imatinib (μM, mean±SEM)viable cell number after 72 hours coincubation with IM (percentage of vehicle control; IM dose: 0.25μM in K562, 0.05μM in KU812)diclofenac (10 μM)ibuprofen (130 μM)no NSAIDsdiclofenac (10 μM)ibuprofen (130 μM)diclofenac (10 μM)ibuprofen (130 μM)K562127%, n=3*48%, n=4**5.7±0.8, n=33.7±0.7, n=3*8.1±0.5, n=3*71.4%, n=3*167%, n=3*KU812122%, n=6*59%, n=6**3.5±0.6, n=32.2±0.4, n=3*6.6±0.8, n=3*64.3%, n=3*145%, n=3*de-novo CP-CML MNC183%, n=50**59%, n=12**1.0±0.1, n=90.7±0.1, n=9*NANANAHealthy donors106%, n=1050%, n=6**NANANANANA*p<0.01,**p<0.001 compared with vehicle controlThe effects of diclofenac and ibuprofen on IM efficacy were then further investigated using primary samples. In keeping with the results observed in cell lines, diclofenac resulted in significant increases in OA, which translated to a significant decrease in IC50imatinib. Ibuprofen induced significant decreases in OA in all patients and healthy donors (Table 1). Conclusion:These finding provide strong evidence for interactions between some NSAIDs and IM which may substantially impact the response of CML patients to IM. While drugs such as diclofenac are likely to have a positive influence on IM efficacy, the findings observed with ibuprofen suggest caution when administrating NSAIDs to CML patients on IM treatment. Disclosures:Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ARIAD: Honoraria, Membership on an entity's Board of Directors or advisory committees. Osborn:Novartis: Research Funding. White:Novartis Pharmaceuticals: Research Funding; BMS: Research Funding.

Proton Pump Inhibitors Augment Nilotinib and Dasatinib Mediated Bcr-Abl Kinase Inhibition

AbstractAbstract 3991Proton pump inhibitors (PPIs) are widely used for the treatment of gastro-oesophageal reflux and peptic ulcer disease. PPIs may not only alter absorption and metabolism of tyrosine kinase inhibitors (TKIs) but could also alter intracellular uptake and retention (IUR) into leukemic cells by interacting with ABCB1 and ABCG2 pump. There is limited literature assessing the interaction of PPI with TKI's at the cellular level. Here we compare the interaction of PPI with dasatinib, imatinib and nilotinib. Mononuclear cells (MNC) of CML-CP patients, K562 and K562-Dox (ABCB1 overexpressing), K562-ABCG2 cells were cultured with 14C-labelled dasatinib, imatinib and nilotinib with or without PPI for 2h and IUR were assessed. The effect of combining PPI with TKI's on Bcr-Abl kinase activity was assessed by measuring p-Crkl (surrogate marker of Bcr-Abl kinase activity). High concentrations of pantoprazole (1 and 2 mM) significantly increased dasatinib IUR in K562-Dox cells (p=0.004 and p=0.0006 respectively) but not in K562 cells (p=0.7 and p=0.1). Similarly, 1 mM (p=0.01) and 2 mM (p=0.007) esomeprazole significantly increased dasatinib IUR in K562-Dox cells but not in K562 cells. These data suggest that high concentration of PPI inhibit ABCB1 mediated dasatinib efflux. This was further supported by significant reduction in IC50dasatinib in K562-Dox cells by 1 mM pantoprazole (112±37 nM to 28±6; p=0.02; n=4). Similarly, 1 mM esomeprazole reduced IC50dasatinib (112±37 nM to 12±3 nM; n=2). This demonstrates that PPI mediated increase in IUR translates to increased kinase inhibition and lower IC50dasatinib. At lower concentrations (100 to 400 μM), neither pantoprazole nor esomeprazole significantly changed the dasatinib IUR or the IC50dasatinib in K562-Dox cells. In K562-ABCG2 cell line 100, 200, 400, 500, 1000 μM pantoprazole reduced IC50dasatinib from 27 nM to 15, 7, 11, 9.5 and 6.5 nM respectively. Similarly, 100, 200, 400, 500 and 1000 μM esomeprazole reduced IC50dasatinib from 21 nM to 15, 11, 8.5, 4.5 and 3.5 nM respectively. These data suggest that PPI enhances dasatinib mediated Bcr-Abl kinase inhibition in ABCG2-overexpressing cells. Although PPI did not change dasatinib IUR significantly in primary CML-MNC (n=10, Table I), it reduced IC50dasatinib (n=4, Table II).Similarly, pantoprazole and esomeprazole (5 to 400 μM) significantly increased nilotinib IUR (n=10, Table I) and significantly reduced IC50nilotinib in CML-MNC (n=3, Table II). Pantoprazole also increased the nilotinib IUR in K562 and K562-Dox cells, and reduced the IC50nilotinib in K562 (500 vs. 250 nM) and K562-Dox (600 vs. 230 nM) cells. Similarly esomeprazole reduced the IC50nilotinib in K562 (500 vs. 250 nM) and K562-Dox (600 vs. 150 nM) cells. The effect of PPI on IC50nilotinib was dose dependent. Pantoprazole and esomeprazole reduced imatinib IUR in K562, K562-Dox and CML-MNC. Pantoprazole increased IC50imatinib in K562 (3.8 to 4 μM) and K562-Dox (6.5 to 8 μM) cells. Similarly, 200 μM of pantoprazole and esomeprazole significantly reduced IM IUR in CML-MNC. However, the effect of pantoprazole on IC50imatinib in CML-MNC was variable and modest (Table II).Our data provide evidence that PPI might interfere with TKI activity. PPI's can enhance the dasatinib and nilotinib mediated Bcr-Abl kinase inhibition in primary CML cells.Table I:Effect of PPI on dasatinib, imatinib and nilotinib intracellular uptake and retention (IUR) in CML-MNC (n=10)TKI at 1μMTKI at 2μMPantoprazole0 μM100μM200μM400μM0uM100μM200μM400μMImatinib13.310.99.98.823.720.418.916.3p-value0.060.010.0040.20.090.01Nilotinib13.016.416.617.430.556.966.274.7p-value0.040.020.030.0040.001<0.001Dasatinib10.311.19.47.918.918.317.718.9p-value0.60.10.080.40.40.2Table II:Effect of PPI on IC50 of nilotinib, dasatinib and imatinib in CML-MNCNilotinibNilotinib + 200 μM pantoprazole (% change)Nilotinib + 200 μM esomeprazole (% change)CML162.5 nM30 nM (−52)55 nM (−12)CML280 nM32 nM (−60)17 nM (−78)CML375 nM68 nM (−9.4)27 nM (−64)DasatinibDasatinib + 200 μM pantoprazole (% change)Dasatinib + 200 μM esomeprazole (% change)CML11.8 nM0.7 nM (−61)1.2 nM (−33)CML23 nM2.6 nM (−13)–CML37 nM2.5 nM (−64)–CML42.25 nM1.7 nM (−25)1.5 nM (−33)ImatinibImatinib + 200 μM pantoprazole (% change)Imatinib + 200 μM esomeprazole (% change)CML11.05 μM0.325 μM (−69)0.8 μM (−23)CML21.8 μM1.35 μM (−25)CML30.83 μM1.15 μM (+38)0.65 μM (−21) Disclosures:Hughes:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. White:Novartis: Honoraria, Research Funding; BMS: Research Funding.

In Contrast to Imatinib, Dasatinib Intracellular Concentration In CML-CD34+ Progenitors Is Not Significantly Different Than That Observed In CD34- Mature Cells.

AbstractAbstract 1205Long term follow up of imatinib (IM) clinical studies and in vitro studies suggest that tyrosine kinase inhibitors (TKI) do not eradicate leukemic stem cells. Refractoriness of leukemic stem cells is postulated to be due to inadequate Bcr-Abl kinase inhibition which in turn could be due to low intracellular uptake and retention (IUR) of TKI. We have previously demonstrated that IM cellular uptake is predominantly mediated by the organic cation transporter protein (OCT-1) and patients with low OCT-1 activity have suboptimal response as compared to patients with high OCT-1 activity. More recently Engler et al (Leukemia 2010) demonstrated that IM IUR is significantly lower in CML-CD34+ cells compared to CD34- cells. This could be due to low expression and activity of the OCT-1 protein and/or high expression of ABCB1 and/or ABCG2 (Jiang et al Leukemia 2007). Although dasatinib is clinically available there are no published data assessing dasatinib IUR in CML-CD34+ progenitors. We and others have previously demonstrated that dasatinib cellular uptake is predominantly OCT-1 independent and dasatinib is a substrate of ABCB1 and ABCG2. We hypothesized that dasatinib IUR would be lower in CML-CD34+ cells compared to CD34- cells. In this study we compare dasatinib and IM IUR; and OCT-1 and ABCB1 mRNA expression in CML-CD34+ and CD34- cells of newly diagnosed CML-CP patients. CD34+ and CD34- cells were incubated with 14C-dasatinib (100 nM and 1 μM) or 14C-IM (2 μM) for 2h and IUR was assessed as described previously (Hiwase et al Clin Cancer Res. 2008). As shown previously, the OCT-1 expression and activity was lower in CML-CD34+ cells, and resulted in lower IM IUR in CML-CD34+ cells compared to CML-CD34- cells (15±5 vs. 27±5; p=0.04; Fig 1A and C). However at a therapeutically achievable concentration (100 nM dasatinib) and at higher concentration (1 μM dasatinib), there was no significant difference in dasatinib IUR in CML-CD34+ and CD34- cells (Fig 1B). Low OCT-1 expression and activity in CML-CD34+ cells did not influence the dasatinib IUR; further confirming that dasatinib cellular influx is predominantly OCT-1 independent. Despite higher ABCB1 mRNA expression in CML-CD34+ cells, the dasatinib IUR was not lower in CML-CD34+ cells compared to CD34- cells. High ABCB1 mRNA expression may not necessarily translate into high ABCB1 activity. To this end, we assessed the effect of PSC-833, an ABCB1 inhibitor, on dasatinib IUR and dasatinib mediated Bcr-Abl kinase inhibition in CML-CD34+ cells. The baseline p-Crkl, a surrogate marker of Bcr-Abl kinase activity, was significantly higher in CML-CD34+ compared to CML-CD34- cells (67±5% vs. 55±8%; p=0.002; n=9). PSC-833 neither increased dasatinib IUR, nor enhanced dasatinib mediated Bcr-Abl kinase inhibition in CML-CD34+ cells (% p-Crkl at 10 nM dasatinib: 20±6 vs. 27±10). Similarly, Ko143, an ABCG2 inhibitor, did not significantly change dasatinib IUR or Bcr-Abl kinase inhibition (% p-Crkl at 10 nM dasatinib: 21±3 vs. 27±10). In summary, although dasatinib is an ABCB1 and ABCG2 substrate, ABCB1 and ABCG2 inhibitors neither increase dasatinib IUR, nor enhance dasatinib mediated Bcr-Abl kinase inhibition in CML-CD34+ cells. This data suggest that dasatinib IUR in CML-CD34+ cells is not influenced by ABCB1 and ABCG2. Hatziieremia et al (Exp. Hematology, 2009) reported that ABCB1 activity is low in CML-CD34+ cells and suggested that it did not influence IM level in CML-CD34+ cells. We further demonstrated that 100 nM dasatinib inhibited ≥95% Bcr-Abl kinase activity in CML-CD34+ and CD34- cells.In summary our data demonstrates that in contrast to IM, the intracellular concentration of dasatinib is equivalent in mature and immature CML cell compartments which may contribute to better targeting of early CML progenitors with dasatinib. [Display omitted] Disclosures:White:Novartis: Honoraria, Research Funding; BMS: Research Funding. Hughes:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.

Chronic Myeloid Leukemia CD34+ cells have reduced uptake of imatinib due to low OCT-1 Activity

Active influx of imatinib in chronic myeloid leukemia (CML) cells is mediated by the organic cation transporter 1 (OCT-1). Functional activity of OCT-1 (OCT-1 Activity) in mononuclear cells is an excellent predictor of molecular response over the first 24 months of imatinib therapy for chronic phase patients. CML progenitor cells are less sensitive to imatinib-induced apoptosis and are likely contributors to disease persistence. We investigated whether alterations in the expression and function of OCT-1 have a role in imatinib resistance in progenitors. We found the intracellular uptake and retention (IUR) of imatinib, OCT-1 Activity and OCT-1 mRNA expression are all significantly lower in CML CD34+ cells compared with mature CD34- cells (P<0.001). However, no differences in IUR or OCT-1 Activity were observed between these subsets in healthy donors. In contrast to OCT-1, ABCB1 and ABCG2 seemed to have no functional role in the transport of imatinib in CML CD34+ cells. Consistent with the observation that nilotinib uptake is not OCT-1 dependent, the IUR of nilotinib did not differ between CML CD34+ and CD34- cells. These results indicate that low imatinib accumulation in primitive CML cells, mediated through reduced OCT-1 Activity may be a critical determinant of long-term disease persistence.

Bosutinib is a Substrate of the ABC Efflux Transporter P-Glycoprotein.

Abstract 3763Poster Board III-699Bosutinib (Bos) (Wyeth Pharmaceuticals) is a dual Src-Abl tyrosine kinase inhibitor developed to inhibit the activity of BCR-ABL in chronic myeloid leukemia. P-glycoprotein (P-gp) is an ATP-binding cassette (ABC) transporter responsible of Imatinib (Im) efflux, and its increased expression can be related to Im resistance. The aim of the study was to define if P-gp is also responsible for the cellular efflux of Bos, and if P-gp altered expression can be related to Bos resistance. Four different K562 Ph+ cell lines were used. 1) K562S, Im sensitive; 2) K562DOX, cells resistant to Doxorubicin that overexpress P-gp (kind gift of JP Marie, Université Pierre et Marie Curie, Paris); 3) K562DOX siP-gp, K562DOX cells carrying a stable silencing of P-gp (kind gift of E Gunsilius, Innsbruck Medical University, Austria); 4) K562DOX H1, K562DOX cells carrying a control vector (kind gift of E Gunsilius). Real Time PCR confirmed that K562DOX and K562DOX H1 express higher levels of P-gp than K562DOX siP-gp (approximately 10 fold) and K562S (approximately 10000 fold). Western blot α-P-gp showed similar results. We then assessed Bos IC50 on the cell lines with a proliferation assay. K562DOX (IC50=175.3nM) and K562DOX H1 (IC50=102 nM) are resistant to Bos if compared to K562DOX siP-gp (IC50=9.1nM) and K562S (IC50=8.7nM). This data confirm that the P-gp overexpression is related to Bos resistance. Using C-14 radiolabeled Bosutinib (C-14 Bos) (Wyeth Pharmaceuticals) we set up an Intracellular Uptake and Retention (IUR) assay on K562S, K562DOX and K562DOX siP-gp cells. Cells were pre-treated for an hour with 0-60μM of Verapamil (Ver), a P-gp inhibitor, and then treated for two hours with 1μM C-14 Bos. In presence of Ver, K562DOX showed a significant increased amount of intracellular C-14 Bos ([] C-14 Bos Ver60μM/ [] C-14 Bos Ver0μM=5.8) that was not observed in K562S or K562DOX siP-gp. These data confirm that the intracellular concentration of Bos is strictly related to the expression levels and to the activity of P-gp. To test the biological effect of P-gp inhibition, we evaluated Bos IC50 by proliferation and apoptosis induction in K562DOX and K562S cells pre-treated with increasing concentrations of Ver. K562S did not show a significant decrease of Bos IC50 even at the highest concentration of Ver (22.5μM). In K562DOX the same concentration of Ver led to a 20 fold decrease of Bos IC50 (9nM), down to a level comparable to Bos IC50 in K562S cells (8.7nM). Thus, the inhibition of P-gp seems to restore the efficacy of Bos on proliferation inhibition and apoptosis induction in BCR-ABL+ cells. Finally, we evaluated the effect of P-gp inhibition on the phosphorylation levels of BCR-ABL with an α-phosphoTyrosine western blot. K562DOX and K562S cells were treated with Bos concentrations ranging from 0 to 80nM, and in absence or presence of Ver (5μM). Phosphorylation levels of BCR-ABL in K562S treated with Ver didn't show any difference when compared to untreated samples. In K562DOX, in absence of Ver, BCR-ABL was phosphorylated even in presence of high Bos concentration; the treatment with Ver restored the sensitivity to Bos, thus leading to a phosphorylation pattern similar to the one of K562S cells. This is compatible with increasing intracellular concentration of Bos after P-gp inhibition which results in increased molecular effect of Bos on BCR-ABL tyrosine phosphorylation. All together, these data confirm the relevance of expression levels and activity of P-gp for the intracellular concentration of Bos. The intracellular concentration of Bos is strictly related to its molecular activity on BCR-ABL and to its biological effects on Ph+ cells. Disclosures:Boschelli:Wyeth Pharmaceuticals: Employment.

Reduced Activity of the OCT-1 Protein in Primitive CML Cells: A Likely Determinant of Stem Cell Resistance in Imatinib Treated CML Patients

Despite cytogenetic and molecular remissions, residual chronic myeloid leukemia (CML) cells persist in the primitive CD34+ compartment in the majority of imatinib treated patients. It has been demonstrated that CD34+ CML cells have a reduced sensitivity to imatinib induced apoptosis. Factors which may contribute to this reduced sensitivity are reduced dependence on BCR-ABL, an increase in BCR-ABL transcripts, increased expression of efflux proteins or decreased expression of drug influx proteins. Our previous studies show that a patient's intrinsic sensitivity to imatinib-induced kinase inhibition (IC50) is related to the intracellular uptake and retention (IUR) of imatinib in peripheral blood mononuclear cells. The organic cation transporter 1 (OCT-1) is the major active influx transporter for imatinib in these cells, and the functional activity of this protein (determined using functional inhibition of OCT-1, in the IUR assay) directly correlates with molecular response to imatinib. In the present study we investigated the role that OCT-1 plays in stem cell resistance to imatinib.Primitive CD34+ and mature CD34− cells were isolated from CML patients and normal individuals using magnetic cell sorting. CML CD34+ cells had a significantly lower IURimatinib than that of CML CD34− cells (Table 1). The addition of the OCT-1 inhibitor, prazosin (100μM), eliminated this difference in IUR (Table 1), indicating that variation in IURimatinib is due to variation in the functional activity of the OCT-1 protein. In addition, the OCT-1 Activity (Table 1) and OCT-1 mRNA expression (expressed as % of BCR: mean CD34+=0.25; CD34−=4.9, p=0.040, n=10) was significantly lower in CML CD34+ cells compared with CML CD34− cells. These differences in IURimatinib and OCT-1 activity between CD34+ and CD34− cells were not observed in normal individuals (Table 1), suggesting this phenomenon is specific to leukemic cells. Furthermore, we isolated the more primitive compartment, CD34+38− cells and less primitive CD34+38+ cells in 4 CML patients. The CD34+38− cells demonstrated a 13% reduction in IURimatinib and a 41% reduction in OCT-1 activity compared with CD34+38+ cells. These data suggest a reduced IUR mediated by low OCT-1 function and/or expression may play a role in the resistance of CML stem cells to imatinib.Table 1: The IUR of 2μM imatinib expressed as ng/200,000 cells (standard deviation)nImatinib IURP value+ PrazosinP valueOCT-1 ActivityP valueCML CD34+1416.05 (4.53)0.00213.02 (3.34)0.5053.25 (2.32)<0.001CML CD34−1427.28 (12.92)14.76 (5.25)14.01 (12.08)Normal CD34+1111.13 (2.66)0.21210.43 (3.80)0.7432.03 (2.09)0.693Normal CD34−1113.92 (5.32)10.93 (3.30)3.52 (5.24)Increased expression of efflux transporters of imatinib (i.e. ABCB1 and ABCG2) has been suggested as an important mechanism for drug resistance. The effect of an ABCB1 inhibitor (PSC833) and ABCG2 inhibitor (Ko143) was assessed in CD34+ cells from 3 CML patients, using the IUR assay. Neither of these drugs had any effect on the IURimatinib in CML CD34+ cells. Additionally the mRNA expression of ABCB1 did not differ between CML CD34+ and CD34− cells (expressed as a % of BCR: mean CD34+=33.7; CD34−=33.77, p=0.064, n=10). These data suggest that alterations in imatinib influx (via OCT-1) are more critical for development of stem cell resistance rather than differences in efflux.We have previously demonstrated that, unlike imatinib, the OCT-1 protein is not involved in nilotinib transport, as the addition of OCT-1 inhibitors does not alter IURnilotinib in patients. Assessing the IUR of nilotinib in CD34+ and CD34− CML cells reveals no significant difference between the two populations (Table 2). Additionally, the IURnilotinib is significantly higher than IURimatinib in CML CD34+ cells (Table 2). In summary, the reduced OCT-1 mediated uptake of imatinib in more primitive, CD34+ CML cells may result in inadequate kinase inhibition and contribute to stem cell resistance in CML. Since nilotinib uptake into CML CD34+ cells is not impaired in the same manner as imatinib, more substantial depletion of the primitive CML cells may be achieved.Table 2: The IUR of 2μM imatinib and nilotinib in the same 11 CML patients. Expressed as ng/200,000 cells (standard deviation)nImatinib IURP valueNilotinib IURP valueP value between imatinib & nilotinib IURCML CD34+1117.80 (5.73)0.00626.35 (7.54)0.2300.007CML CD34−1130.28 (12.33)22.05 (8.68)0.076

Enhancing the Functional Activity of the OCT-1 Influx Pump May Overcome the Negative Impact of Low OCT-1 Activity in Imatinib Treated CML Patients

The human organic cation transporter-1 (hOCT-1) is the major active influx protein responsible for the transport of imatinib into blood cells 1,2. The functional activity of the OCT-1 protein is defined as the intracellular uptake and retention (IUR) of 14-C labelled imatinib into patient pre therapy mononuclear blood cells over a two hour period, which is inhibited by OCT-1 inhibitors such as prazosin or procainamide. The level of OCT-1 activity is a key determinant of the interpatient variation observed in intrinsic sensitivity to imatinib induced kinase inhibition (IC50imatinib3). We have previously demonstrated that a significantly greater proportion of de-novo CML patients with high functional activity of OCT-1, achieve a major molecular response (MMR: 3 log reduction in BCR-ABL mRNA from standardised baseline) when treated with imatinib, than patients with low OCT-1 Activity 4. We have also identified a link between dose and OCT-1 Activity, demonstrating that the negative impact of low OCT-1 Activity could be overcome to a variable extent by imatinib dose increase. However, not all patients can dose increase, largely because of tolerability issues. While the transport of second-generation ABL-kinase inhibitors (nilotinib and dasatinib5) is not OCT-1 mediated, the long term effect of these drugs is not yet known. Hence, we sought to identify strategies to increase OCT-1-mediated imatinib uptake. We queried the drug gene expression signatures in version 1 of the Connectivity Map (CMAP; Lamb J, Nat. Rev. Cancer 7; 54–60, 2007: http://www.broad.mit.edu/cmap) with 3 transporters including OCT-1. This identified the Rho kinase inhibitor fasudil and COX-2 inhibitor / celecoxib analogue LM1685 as potential up-regulators of OCT-1 mRNA. The impact of these drugs on OCT-1 mRNA expression and IC50imatinib (fasudil alone to date) has been analysed in two bcr-abl positive cell lines (K562 and KU812). The effect of these two candidate OCT-1 enhancers on OCT-1-mediated imatinib uptake was also assessed in 10 newly diagnosed chronic phase CML patients, previously demonstrated to have low OCT-1 Activity (4 with no demonstrable OCT-1 Activity), using the IUR assay.Table 1: Assessing the effects of fasudil and LM1685 on the intracellular transport of imatinib. These data demonstrate a statistically significant increase in OCT-1 Activity with LM1685, and show a strong trend towards significance with fasudil. Importantly, we show that patients with no demonstrable OCT-1 Activity (0ng/200,000 cells) have detectable Activity in the presence of both fasudil (Range 1.5 to 2ng/200,000 cells) and LM1685 (Range 1.5 to 4.5 ng/200,000 cells). We have previously demonstrated that patients with no detectable OCT-1 Activity universally fail to achieve imatinib therapeutic response milestones (imatinib failure), whereas 54% of patients with low, but detectable OCT-1 Activity achieve these milestones4. The ability to enhance the functional activity of the OCT-1 protein may therefore be of significant clinical relevance in this group. In addition we demonstrate an increase in imatinib IUR which, along with the increase in OCT-1 Activity, is likely associated with increased OCT-1 mRNA levels. Significantly, in the two CML cell lines tested we show a marked reduction in the IC50imatinib indicating that the observed increase in IUR and OCT-1 Activity translates to an increase in the kinase inhibitory activity of imatinib. Preliminary analysis in one patient analysed to date also indicates a reduction in IC50 from 0.48 to 0.35μM in the presence of fasudil. In the clinical scenario the use of such OCT-1 enhancers may improve the response of some imatinib treated patients to both standard and increased dose imatinib. Importantly, these findings validate the use of resources such as C-MAP to identify candidate drugs that may mediate desired changes in the levels of key proteins resulting in improved response to therapy.Fasudil (10μM) %increase from controlLM1685 (1μM) % increase from controlIUR of imatinibOCT-1 ActivityIC50imatinibIUR of imatinibOCT-1 ActivityK56276% (n=5)163% (n=5)51% reduction (n=3)41% (n=2)122% (n=2)KU81210.7% (n=2)75% (n=2)15% reduction (n=2)NANACML patients n=108%89%9%114%p value>0.050.08>0.050.03

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.

Interaction of Imatinib with Human Organic Ion Carriers

The activity of imatinib in leukemia has recently been linked with expression of the organic cation transporter 1 (OCT1) gene SLC22A1. Here, we characterized the contribution of solute carriers to imatinib transport in an effort to further understand mechanisms involved in the intracellular uptake and retention (IUR) of the drug. IUR of [3H]imatinib was studied in Xenopus laevis oocytes and HEK293 cells expressing OATP1A2, OATP1B1, OATP1B3, OCT1-3, OCTN1-2, or OAT1-3. Gene expression was determined in nine leukemia cell lines using the Affymetrix U133 array. Imatinib was not found to be a substrate for OCT1 in oocytes (P = 0.21), whereas in HEK293 cells IUR was increased by only 1.20-fold relative to control cells (P = 0.002). Furthermore, in 74 cancer patients, the oral clearance of imatinib was not significantly altered in individuals carrying reduced-function variants in SLC22A1 (P = 0.99). Microarray analysis indicated that SLC22A1 was interrelated with gene expression of various transporters, including ABCB1, ABCC4, ABCG2 (negative), and OATP1A2 (positive). Imatinib was confirmed to be a substrate for the three efflux transporters (P < 0.05) as well as for OATP1A2 (P = 0.0001). This study suggests that SLC22A1 expression is a composite surrogate for expression of various transporters relevant to imatinib IUR. This observation provides a mechanistic explanation for previous studies that have linked SLC22A1 with the antitumor activity of imatinib. Because of its high expression in the intestine, ciliary body, gliomas, and leukemia cells, OATP1A2 may play a key role in imatinib pharmacokinetics-pharmacodynamics.

Most CML patients who have a suboptimal response to imatinib have low OCT-1 activity: higher doses of imatinib may overcome the negative impact of low OCT-1 activity

AbstractInterpatient variability in intracellular uptake and retention (IUR) of imatinib may be due to variable function of the OCT-1 influx pump. OCT-1 activity was measured in pretherapy blood from chronic myeloid leukemia (CML) patients by calculating the difference in IUR of [14C]-imatinib with and without OCT-1 inhibition. Of patients with higher than median (high) OCT-1 activity, 85% achieved major molecular response (MMR) by 24 months, versus 45% with no more than a median (low) OCT-1 activity. Assessing patients receiving 600 mg imatinib per day and those averaging fewer than 600 mg over 12 months of therapy revealed patients with high OCT-1 activity achieved excellent molecular response regardless of dose, whereas response of patients with low OCT-1 activity was highly dose dependent. Of patients with low OCT-1 activity who received fewer than 600 mg, 45% failed to achieve a 2-log reduction by 12 months, and 82% failed to achieve a MMR by 18 months, compared with 8% and 17% in the cohort with high OCT-1 activity and dose less than 600 mg/day (P = .017 and P = .022). OCT-1 activity is an important determinant of molecular response to imatinib, with predictive value closely linked to dose. This pretherapy assay identifies patients at greatest risk of suboptimal response where dose intensity is critical, and those likely to respond equally well to standard dose imatinib.

Molecular Response to Imatinib Is Dependent on Dose in CML Patients with Low OCT-1 Influx Activity. Patients with High Activity May Respond Equally Well to Standard or Increased Dose Imatinib.

We have shown that IC50 is predictive of molecular response in newly diagnosed CML patients treated with 600mg of imatinib (TIDEL). OCT-1 is an influx transporter for imatinib and key determinant of interpatient variation in the IC50 assay. Using 14C imatinib we have performed an intracellular uptake and retention (IUR) assay and RQ-PCR for OCT-1 mRNA expression on blood cells from 42 patients at diagnosis, enrolled to TIDEL. Using the OCT-1 inhibitor Prazosin, we determined OCT-1 in-vitro activity to be the difference in IUR between assays performed with and without Prazosin. The median OCT-1 activity was 6.1ng (range of -3.6 to 27ng). Grouping patients about the median, into low and high OCT-1activity, we found a significant difference between the two groups in molecular response.Log Reduction BCR-ABL based on OCT-1 ActivityMonths6M12M18M24MLow OCT-1 Activity n=191.92.42.62.5High OCT-1 Activity n=232.072.93.63.8p value0.050.050.0030.007Low OCT-1 Activity<600mg n=101.41.71.72.4600mg n=92.72.62.93.6p value0.010.0230.0460.042High OCT-1 Activity<600mg n=112.42.93.33.7600mg n=122.42.83.33.9p value0.630.690.820.66Dividing groups into patients receiving 600mg imatinib and those receiving <600mg over the first 12 months of therapy revealed patients with high OCT-1 activity generally achieved excellent molecular response regardless of dose, whereas the response of patients with low OCT-1 activity was highly dose dependent. There was a significant difference in molecular response between the low and high OCT-1 activity groups on <600 mg (p=0.004 at 24 months) but no difference in the 600mg cohort, further indicating dose sensitivity of low OCT-1 activity patients. The level of OCT-1 mRNA (n=30) as determined by RQ-PCR correlated with the in-vitro OCT-1 activity (r=0.381 p=0.04). However, in this smaller cohort, OCT-1 mRNA level was not predictive of molecular response (low OCT-1 3.5 log(n=15); high OCT-1 3.9 log(n=16) p=0.169). The IC50 is predictive of molecular response to 12 months (low IC50 log reduction 3.0 (n=20) high IC50 2.5 (n=15) p=0.02), but not in this smaller cohort to 24 months (p=0.15). TIDEL mandated dose increase to 800mg where tolerable. We assessed the log reduction from 12 to 24 months based on dose.Log reduction from 12 to 24 months.Dose increaseYesNop valueLow OCT-1 activity0.33 n=120 n=70.002High OCT-1 activity0.59 n=140.46 n=90.637p value0.7030.034There was no statistical difference between the low and high OCT-1 activity groups when dose was escalated, however with constant dose, patients with low OCT-1 activity performed less well (Table 2). We conclude that OCT-1 influx activity is an important determinant of molecular response to imatinib, but its predictive value is closely linked to the dose received. This functional assay of OCT-1 activity performed at diagnosis may identify patients most likely to benefit from higher doses of imatinib or second generation ABL kinase inhibitors, and a cohort who are likely to respond well to standard dose imatinib.