OCT-1 Activity Research Articles

Oct1 Is a Switchable, Bipotential Stabilizer of Repressed and Inducible Transcriptional States

Little is known regarding how the Oct1 transcription factor regulates target gene expression. Using murine fibroblasts and two target genes, Polr2a and Ahcy, we show that Oct1 recruits the Jmjd1a/KDM3A lysine demethylase to catalyze the removal of the inhibitory histone H3K9 dimethyl mark and block repression. Using purified murine T cells and the Il2 target locus, and a colon cancer cell line and the Cdx2 target locus, we show that Oct1 recruits the NuRD chromatin-remodeling complex to promote a repressed state, but in a regulated manner can switch to a different capacity and mediate Jmjd1a recruitment to block repression. These findings indicate that Oct1 maintains repression through a mechanism involving NuRD and maintains poised gene expression states through an antirepression mechanism involving Jmjd1a. We propose that, rather than acting as a primary trigger of gene activation or repression, Oct1 is a switchable stabilizer of repressed and inducible states.

The pharmacogenetics of metformin and its impact on plasma metformin steady-state levels and glycosylated hemoglobin A1c

The aim of this study was to evaluate the effect of genetic variations in OCT1, OCT2, MATE1, MATE 2, and PMAT on the trough steady-state plasma concentration of metformin and hemoglobin A1c (Hb1Ac). The South Danish Diabetes Study was a 2 x 2 x 2 factorial, prospective, randomized, double-blind, placebo-controlled, multicentre study. One hundred and fifty-nine patients received 1 g of metformin, twice daily continuously, and 415 repeated plasma metformin measurements were obtained after 3, 6, and 9 months of treatment. The mean trough steady-state metformin plasma concentration was estimated to be 576 ng/ml (range, 54–4133 ng/ml, p = 0.55) and correlated to the number of reduced function alleles in OCT1 (none, one or two: 642, 542, 397 ng/ml; P = 0.001). The absolute decrease in Hb1Ac both initially and long term was also correlated to the number of reduced function alleles in OCT1 resulting in diminished pharmacodynamic effect of metformin after 6 and 24 months. In a large cohort of type 2 diabetics, we either confirm or show for the first time: (a) an enormous (80-fold) variability in trough steady-state metformin plasma concentration, (b) OCT1 activity affects metformin steady-state pharmacokinetics, and (c) OCT1 genotype has a bearing on HbA1c during metformin treatment.

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.

Early Switching From Imatinib to Nilotinib In CML Patients Failing to Achieve Early Molecular Targets May Not Be An Effective Approach In Patients with Very Low OCT-1 Activity: A TIDEL II Sub-Study

AbstractAbstract 356We have previously demonstrated that CML patients with very low OCT-1 activity (Quartile 1 (Q1)<4 ng/200,000 cells) have significantly poorer outcomes with respect to response, mutation development, progression, and survival than patients with higher OCT-1 activity(OA). We have also demonstrated that the influx pump OCT-1 is associated with the active uptake of imatinib, but not nilotinib (NIL), into target CML cells. Hence, if the prognostic significance of OA primarily reflects IM transport we predicted that patients with very low OA who had poor initial responses to imatinib, should respond well when switched to NIL. In the Tidel II study CML-CP patients are treated with 600mg/day imatinib (IM) upfront and are dose escalated to 400mg BID or switched to NIL 400mg BID for either intolerance (Grade III/IV or persistent Grade II non-haematological toxicity) or failure to meet pre defined molecular targets. These targets are early markers of sub-optimal response, but not IM failure: failure to achieve <10% BCR-ABL IS by 3 months, <1% by 6 months and <0.1% by 12months. Hence this study is designed to enable early efficacious intervention in the case of poor response, to prevent frank therapeutic failure. In this study we examine the cohort of patients who have switched to NIL and compare their response on NIL to that achieved on IM. To date 63 patients on this trial have greater than 12 month molecular follow up, and OA quantified. 26/63 patients have OA<4ng/200,000 cells. As demonstrated in Table 1, and in keeping with previous findings, patients with low OA have significantly poorer rates of MMR by 12 months, and higher rates of study discontinuation when compared to patients with ≥4.0ng/200,000 (high OA). Importantly the study design of Tidel II has enabled the more careful assessment of therapeutic intervention. Of note 23% of patients with low OA had a dose increase and 42% switched to nilotinib because of failure to meet pre-stated milestones. This compares with only 8% of remaining patients being dose increased and 14% switching to NIL(p=0.007).Table 1:Showing the response of patients within the two OCT-1 Activity Groups.MMR by 12 moMutationsOff StudyIncreased dose 400mg BID IMMMR on 400mg BID IMSwitched to NILMMR on NIL<4.0ng/200,000 cells n=2637%19%19%23%16.6%42%10%≥4.0ng/200,000 cells n=3781%0%2.7%8.1%100%13.5%100%p=<0.0010.0060.0370.1410.050.0070.007With a median follow up after switching to NIL of 12 months (Low OA R=6-21 high OA R=9-12 p=0.887) all patients with high OA, not previously in MMR (n=4), achieved MMR within 6 months. In contrast 1/10 low OA patients not previously in MMR achieved MMR on NIL. Interestingly, not all patients with low OA were switched to NIL because of sub-optimal IM response. Four of this cohort were intolerant to 600mg IM (one of these 4 achieved MMR), while all patients in the high OA cohort were switched because of intolerance. This implies that a low OA is not overcome with early switch to NIL, despite NIL transport being unrelated to OCT-1. This may be due to other intrinsic factors, possibly related to leukaemia biology and majority cell type for which the OA assay may be a surrogate measure. Alternatively, the poor response to imatinib in low OA patients has facilitated the activation of other resistance mechanisms, that in turn lead to a poor response after switching implying that patients with low OA may require upfront treatment with a TKI that is OCT-1 independent. Importantly only 1 of the patients on NIL had a mutation prior to switching, with another patient transforming to blast crisis with a T315I mutation while on NIL. Thus the presence of kinase domain mutations does not explain these findings. Conclusions:Low OA is associated with poor response to IM. Here we show that the MMR rate for patients with low OA was not substantially improved by the TIDEL II treatment strategy. This is despite the protocol allowing for early dose escalation or switch for suboptimal response. This preliminary data suggests that patients with low OA may have more generalized insensitivity to kinase inhibitor therapy which is not simply related to drug transport. These are important findings in the setting of multiple TKIs being available, and suggest that in patients with very low OA a regimen of selective TKI intensification may not be the optimal approach. Disclosures:White:Novartis: Honoraria, Research Funding; BMS: Research Funding. Slader:Novartis Pharmaceuticals: Employment, Equity Ownership. Osborn:Novartis Pharmaceuticals: Research Funding; Bristol Myers Squibb: Research Funding. Mills:Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hughes:Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria.

OCT-1 function varies with cell lineage but is not influenced by BCR-ABL

Despite the excellent responses to imatinib therapy observed in patients with chronic phase chronic myeloid leukemia, approximately 25% of patients display primary resistance or suboptimal response. The OCT-1 activity in mononuclear cells reflects the efficiency of active influx of imatinib. OCT-1 activity in mononuclear cells is highly variable between patients and significantly correlates with a patient's molecular response to imatinib treatment and overall survival. The present study examined whether cell lineage and BCR-ABL expression influenced OCT-1 activity. The OCT-1 activity and OCT-1 mRNA expression was assessed in pure populations of neutrophils, monocytes and lymphocytes recovered from chronic myeloid leukemia patients at diagnosis, in cytogenetic remission and normal individuals. The role of BCR-ABL on OCT-1 activity and differentiation was examined in a cell line model of ectopic BCR-ABL expression. The OCT-1 activity and OCT-1 mRNA expression was highest in the neutrophil population and lowest in lymphocytes (P<0.05). This was observed for patients at diagnosis, in cytogenetic remission and normal individuals. Interestingly, neutrophil OCT-1 activity was not significantly different between patients at diagnosis, in remission and normal donors. This was also observed for monocytes and lymphocytes. Furthermore, OCT-1 activity in mononuclear cells was significantly correlated with the OCT-1 activity in neutrophils (P=0.001). In a cell line model in which BCR-ABL was ectopically expressed, we found no evidence that BCR-ABL directly affected OCT-1 expression and function. However, BCR-ABL stimulated granulocyte differentiation which, in turn, led to significantly increased OCT-1 activity (P=0.024). These studies suggest that the predictive OCT-1 activity in patient mononuclear cells is strongly related to cell lineage, particularly the presence of neutrophils in the peripheral blood. Furthermore, BCR-ABL expression is unlikely to directly influence OCT-1 activity but may have an indirect role by enhancing granulocyte differentiation.

The poor response to imatinib observed in CML patients with low OCT-1 activity is not attributable to lower uptake of imatinib into their CD34+ cells

AbstractThe functional activity of the organic cation transporter 1 (OCT-1) protein in chronic myeloid leukemia (CML) mononuclear cells (MNCs) is highly predictive of molecular response in imatinib treated patients. Here we investigate whether the MNC OCT-1 activity (OA) provides a surrogate indicator of effective targeting of the more immature CD34+ cells. While confirming our previous findings that high MNC OA is significantly associated with the achievement of major molecular response (MMR; P = .017), the present studies found no relationship between high CD34+ OA and the achievement of MMR. Furthermore, no correlation was found between the MNC OA and the CD34+ OA in matched CML samples. These results suggest that the predictive value of the MNC OA may primarily reflect the effective targeting and subsequent reduction of mature CML cells. Therefore kinase inhibition in these mature cells, and not the CD34+ cells, may be the key determinant of response in CML.

OCT-1 activity measurement provides a superior imatinib response predictor than screening for single-nucleotide polymorphisms of OCT-1

OCT-1 activity measurement provides a superior imatinib response predictor than screening for single-nucleotide polymorphisms of OCT-1

Functional Activity of the OCT-1 Protein Is Predictive of Long-Term Outcome in Patients With Chronic-Phase Chronic Myeloid Leukemia Treated With Imatinib

Organic cation transporter-1 (OCT-1) activity (OA), a measure of the OCT-1-mediated influx of imatinib into CML mononuclear cells (MNCs), is predictive of major molecular response (MMR) at 12 and 24 months in patients with untreated CML. We now report the impact of OA on loss of response, disease transformation, and survival after 5 years of imatinib. OA is defined as the difference in intracellular concentration of carbon-14-imatinib with and without OCT-1 inhibition. OA was measured in blood from 56 patients with untreated chronic-phase CML. More patients who had high OA (ie, > median OA value) achieved MMR by 60 months compared with patients who had low OA (89% v 55%; P = .007). A low OA was associated with a significantly lower overall survival (87% v 96%; P = .028) and event-free survival (EFS; 48% v 74%; P = .03) as well as a higher kinase domain mutation rate (21% v 4%; P = .047). These differences were highly significant in patients who averaged less than 600 mg/d of imatinib in the first 12 months but were not significant in patients averaging >/= 600 mg/d. Patients with very low OA (ie, quartile 1) were the only group who developed leukemic transformation (21% in quartile 1 v 0% in all other quartiles; P = .002). Measurement of OA pretherapy is a predictor for the long-term risk of resistance and transformation in patients with imatinib-treated CML. Early dose-intensity may reduce the negative prognostic impact of low OA. We propose that OA could be used to individualize dosage strategies for patients with CML to maximize molecular response and optimize long-term outcome.

Abstract 3538: An atomic absorption spectroscopy assay (AASA) as a surrogate method to measure imatinib (IM) uptake via the organic cation transporter 1 (OCT1)

Abstract Background. In chronic myeloid leukaemia (CML) 15 to 30% of patients (pts) treated with the ABL specific tyrosine kinase inhibitor IM may develop resistance. In these pts clonal evolution, amplification of the BCR-ABL gene or kinase domain mutations have been identified as underlying mechanisms. Additionally, pharmacokinetic alterations leading to reduced intracellular imatinib levels may also exist. IM is transported into the cell via the OCT1. The activity of this transporter could play a pivotal role for the intracellular IM concentration. Until now, uptake efficacy of IM is primarily measured by using radioactive labelled IM. Due to the limitations of these methods, we tried to establish alternative methods to measure the activity of OCT1 using cellular OCT1 dependent oxaliplatin (OX) uptake as a surrogate. Materials &amp; Methods. To quantitatively analyse OX uptake in human cell lines an AASA was established. Conversely, radioactive IM uptake was measured by scintillation analysis. We compared the uptake of OX and IM in different human cell lines, including JURKAT, Molt-4, K-562, KU-812, Lama-87, KCL-22 and OCI-AML-2. Ten different transport inhibitors were used to block IM or OX uptake, respectively. Furthermore, we investigated the expression levels of different influx (OCT1, OCT2, OCT3) and efflux transporters (ABCB1, ABCC1, ABCG2) in those cell lines. Results. IM and OX both were taken up linearly into cells within a range from 0,4 to 2 µM (IM) or 1 to 10 µM (OX). 150 mM verapamil and 100 µM prazosin could effectively inhibit cellular uptake of both drugs (21%±8% and 27%±8%). However, a differential uptake pattern between OX and IM was observed in relation to the different cell lines. In contrast, we could show that 15 µM citalopram inhibited the transport of 5 µM OX in a similar type as the uptake of 1-2 µM IM into all cell lines tested yet (1,14±0,15*). In this regard citalopram may be most useful for further studies on OCT1 activity. Using real-time PCR, an adequate expression of OCT1 mRNA could be detected in all cell lines tested, albeit the levels differed significantly from 150 to 27.000 fold less than in the internal control (ß-actin). However, the mRNA expression level of OCT1 does not seem to be a predictor for IM or OX uptake. The expression pattern of the efflux transporters showed no significant distribution, too. Conclusions. Here, we demonstrate the feasibility and accuracy of AASA testing of oxaliplatin as a surrogate marker for IM uptake via OCT1. As this methodology is secure, fast and sensitive, this approach may substitute conventional testing of OCT1 activity by radio labelled IM. On a clinical level, this procedure could be helpful as a clinical tool to test IM pharmacokinetics in CML patients. However, patient sample derived data will be needed to further evaluate this approach. * 1 would stand for 100% equality of uptake of imatinib or oxaliplatin Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3538.

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.

Nilotinib does not significantly reduce imatinib OCT-1 activity in either cell lines or primary CML cells

Nilotinib does not significantly reduce imatinib OCT-1 activity in either cell lines or primary CML cells

OCT-1 Activity in CML CD34+ Cells Is Not Predictive of Molecular Response to Imatinib Treatment in CP-CML Patients, Despite the Strong Predictive Value of MNC OCT-1 Activity.

Abstract 2189Poster Board II-166 Introduction.The organic cation transporter 1 (OCT-1) is the major active influx transporter for imatinib in PB mononuclear cells (MNC) from CML patients. We have previously demonstrated that the functional activity of the OCT-1 protein (OCT-1 Activity, OA) in MNC from chronic phase (CP) CML patients is highly variable (mean: 7.2 ng/200,000 cells, standard deviation: 6.4). We also demonstrated that the MNC OA is significantly related to a patient's sensitivity to imatinib-induced kinase inhibition, and their subsequent molecular response. In addition, we have recently demonstrated that OA is predictive of event free and transformation free survival to 5 years (White ASH submitted 2009). This is of a greater importance for those patients on lower doses of imatinib (below 600mg). For these patients who have a low MNC OA, 82% failed to achieve a major molecular response (MMR) by 18 months of imatinib treatment, as opposed to 17% for patients with a high MNC OA (p=0.022). Recently, we have shown that the OA in CML CD34+ cells is significantly lower than that found in mature CML cells (mean CD34+: 3.9, CD34-: 11.6 ng/200,000 cells, p<0.001, n=36), however interpatient variation in CD34+ OA still remains (standard deviation: 2.65). Given the importance of low MNC OA as a prognostic indicator of poor molecular response, we sought to ascertain whether a patient's MNC OA was related to the OA in their CD34+ cells, and as such could be used as a surrogate indicator for efficient targeting of the primitive population. To do this, we firstly looked for a correlation between the OA in a patient's MNC and their CD34+ cells. Secondly, we assessed the predictive value of CD34+ OA for MMR in imatinib treated patients. Methods.MNC and CD34+ cells were isolated from imatinib naïve, newly diagnosed CP-CML patients using density gradient centrifugation and magnetic cell sorting, respectively. OA was determined using [14C]-labeled imatinib and the OCT-1 inhibitor prazosin. OA is calculated as the difference between the uptake and retention of imatinib in the absence and presence of prazosin. Statistical analyses were performed using the Mann-Whitney Rank Sum or Student's t-test, correlations were performed using the Spearman-Rank Order. Results.In 34 CML patients no correlation was observed between the OA in their MNC and the OA in their CD34+ cells (R=0.09, p=0.577). Furthermore, when these patients were divided as having high or low OA in their MNC (about a previously described median of 7.2 ng/200,000 cells), no difference was seen between the OA in their corresponding CD34+ cells (Table 1).Table 1The mean OCT-1 Activity (OA) in a patient's CD34+ cells expressed as ng/200,000 cells (Standard Deviation)nCD34+ OAP ValueLow MNC OA254.02 (2.78)0.854High MNC OA93.83 (2.53)Finally, in 19 patients where 12 month response data was available, patients were grouped according to the achievement or not of MMR by 12 months. MNC OA was found to be significantly associated with the achievement of MMR (Table 2). This suggests that patients with a high OA in their MNC achieve better molecular responses, which is in agreement with our previous findings. However, assessment of CD34+ OA failed to demonstrate a relationship between OA and the achievement of MMR (Table 2). Therefore, a low OA in patient's CD34+ cells (unlike MNC) does not appear to be associated with poor molecular responses.Table 2The mean OCT-1 Activity (OA) in patients MNC and CD34+ cells expressed as ng/200,000 cells (Standard Deviation)nMNC OAP ValueCD34+ OAP ValueNo MMR73.13 (2.90)0.0313.65 (2.29)0.979Yes MMR128.38 (5.44)3.63 (1.81) Conclusion.No relationship was identified between the OA measured in a patient's MNC and that in their CD34+ cells. Furthermore, CD34+ OA is not predictive of a patient's molecular response to imatinib treatment. This is in contrast to MNC OA, which is predictive of event free and transformation free survival to 5 years of imatinib treatment. This data suggests that the predictive value of the MNC OA may primarily reflect the effective targeting and subsequent eradication of mature CML cells. And that kinase inhibition in these mature cells (as opposed to CD34+ progenitor cells) may be the key determinant of MMR achievement in CP-CML.Supported by Novartis Oncology, Clinical Development, TOPS Correlative Studies Network Disclosures:Zannettino:Novartis Pharmaceuticals: Research Funding. White:Novartis Pharmaceuticals: Honoraria, Research Funding. Hughes:Novartis Pharmaceuticals: Honoraria, Research Funding, Speakers Bureau.

The Functional Activity of the OCT-1 Protein Is Predictive of Molecular Response and Survival in CP-CML Patients Treated with Imatinib: A 5 Year Update of the TIDEL Trial.

Abstract 507The major active influx protein for imatinib into target BCR-ABL positive cells is the organic cation transporter OCT-1. We have previously demonstrated that the functional activity of the OCT-1 protein (OCT-1 activity) is predictive of molecular response in TIDEL (trial of imatinib 600 mg/day with selective dose intensification in untreated CP-CML) The OCT-1 activity (OA) is measured in mononuclear cells from untreated CML patients by calculating the intracellular concentration of 14-C imatinib less the intracellular concentration in the presence of OCT-1 inhibition. To address the question of whether OA is predicting only the rate of response, we now investigate the impact of OA on response and progression at 5 years. There is a significant difference in the achievement of MMR (p=0.007) and CMR by 60 months (p=0.032) (Table 1). Six patients developed kinase domain mutations over the course of this study. 5/6 had low OA. Significantly, for the first time addressing Event Free Survival (events defined as loss of CHR, MCR or CCR, progression to AP or BC or change of therapy due to unsatisfactory efficacy), we demonstrate that more patients with high OA are event free at 5 years when compared to patients with low OA (Table 1).Table 160 month analysis of the % of patients achieving MMR, CMR and EFS rates.The % of patients achieving:- By 60 monthsMMRCMREFSMutationsOverall (n=56)71506412Low OA 0-7.2 ng/200,000 cells (n=29)55345221High OA >7.2 ng/200,000 cells (n=27)8967784p-value0.0070.0320.0380.047To determine whether the detrimental effect of low OA on survival was more significant in those patients with OA in the lowest quartile (Q1) we compared the response of Q1 patients to all other patients (Table 2). These data demonstrate importantly, that patients in Q1 have significantly poorer outcomes, than the remainder of the patient cohort.Table 2Overall, Event and Progression Free Survival in Q1 compared to the remainder of the cohort.Event Free SurvivalTransformation Free SurvivalOverall SurvivalQ1 (<4.0ng/200,000cells: n=14)437979Remainder (n=42)7110098p-value0.0230.0020.009In previous analyses we have shown that the effects of a low OA can be partially overcome by higher imatinib doses. Limiting the analyses to those patients receiving <600mg average daily dose over the first 12 months there was a significant difference in the achievement of MMR (low OA (n=11) 27%: high OA (n=12) 92% p=0.021) and EFS (36% vs 75% p=0.03). In patients receiving ≥600 mg there was no significant difference between the groups, reinforcing the importance of dose.In 45 patients we examined the expression of OCT-1 mRNA for prediction of MMR, CMR, EFS and mutation development. Dividing the patients into low and high OCT-1 expression about the median we found that the level of mRNA is not predictive of MMR (low–60% vs high 78 p=0.241) CMR (low–45% vs high 55 p=0.456) EFS (low–55% vs high 70 p=0.315) or mutation development (low–18% vs high 14% p=0.666). These data indicate that the level of OCT-1 mRNA is not sufficiently discriminating to predict response and progression.While our previous studies demonstrated that OA could predict the rate of decline in BCR-ABL over the first 12-24 months, this update demonstrates for the first time, that this assay can identify nearly all patients (>80%) who fail to achieve MMR in the long term. Most importantly OA is also strongly predictive of resistance and progression events. Functional assessment of OCT-1 Activity provides prognostic information that is more discriminating than assaying the level of OCT-1 mRNA. This long term study reinforces the notion that OA is an important predictive variable in CP-CML patients treated with IM. It provides further evidence that OA is a critical variable to consider in future trials of imatinib and a key factor to enable individualization of imatinib dose to optimize the long term outcome for CML patients. Disclosures:White:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding. Manley:Novartis: 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.

Clinical Strategies to Achieve an Early and Successful Response to Tyrosine Kinase Inhibitor Therapy

Imatinib is the standard of care for previously untreated chronic myeloid leukemia (CML), with high response rates that lead to improved event-free and overall survival compared with interferon alfa. Imatinib dose is one important factor affecting response, and early clinical studies showed promising molecular response rates with high-dose therapy. Large, randomized trials are now ongoing to test this potential benefit and establish whether a starting dose of 800 mg/d improves long-term clinical outcomes compared with the current standard dose of 400 mg/d. Low plasma imatinib levels are associated with a decreased chance of response. The importance of imatinib dosing and plasma levels is likely due to their impact on intracellular concentrations of the drug. Cellular influx of imatinib is mediated by the OCT-1 protein, and patients with low OCT-1 activity may benefit from dose-intensive therapy. For nonresponding or slowly responding patients, dose escalation to 600 to 800 mg/d may lead to durable responses in patients with primary or secondary resistance. Regular monitoring of response is crucial to maximize therapeutic success, and improved understanding of the factors affecting response will guide future clinical strategies.

BOB.1 of the channel catfish, Ictalurus punctatus: Not a transcriptional coactivator?

Expression of the immunoglobulin heavy chain ( IGH) locus of the channel catfish ( Ictalurus punctatus) is driven by the Eμ3′ enhancer, whose core region contains two octamer motifs and a μE5 site. Orthologues of the Oct1 and Oct2 transcription factors have been cloned in the channel catfish and shown to bind to the octamer motifs within the core enhancer. While catfish Oct2 is an activator of transcription, catfish Oct1 failed to drive transcription and may act as a negative regulator of IGH transcription. In mammals, the Oct co-activator BOB.1 (B cell Oct-binding protein1, also known as OCA-B and OBF-1) greatly enhances the transcriptional activity of Oct factors and plays an important role in the development of the immune system. An orthologue of BOB.1 has been cloned in the catfish, and its function characterized. The POU binding domain of the catfish BOB.1 was found to be 95% identical at the amino acid level with the binding domain of human BOB.1, and all the residues directly involved in binding to the Oct-DNA complex were conserved. Despite this conservation, catfish BOB.1 failed to enhance transcriptional activation mediated by endogenous or co-transfected catfish Oct2, and failed to rescue the activity of the inactive catfish Oct1. Electrophoretic mobility shift assays showed that catfish BOB.1 was capable of binding both catfish Oct1 and Oct2 when they formed a complex with the Oct motif. Analysis of recombinant chimeric catfish and human BOB.1 proteins demonstrated that the failure to drive transcription was due to the lack of a functional activation domain within the catfish BOB.1.

CML Patients with Low OCT-1 Activity Achieve Better Molecular Responses on High Dose Imatinib Than on Standard Dose. Those with High OCT-1 Activity Have Excellent Responses on Either Dose: A TOPS Correlative Study

We have previously demonstrated in CML patients enrolled to the Australian TIDEL trial, (600mg imatinib upfront in newly diagnosed patients) that patients with high OCT-1 activity, measured in patient blood mononuclear cells prior to imatinib start, achieve a superior molecular response, compared to those with low OCT-1 activity 1. Furthermore, the impact of low OCT-1 activity could be partially overcome with increased imatinib dose. We now prospectively test the predictive value of OCT-1 activity on the achievement of a major molecular response (<0.1 BCR-ABL IS) by 12 months, in CML patients enrolled to the TOPS trial (randomised 400 vs 800 mg imatinib). A subset of 131 TOPS2 patients had OCT-1 activity measured prior to the start of therapy, as part of the Global Novartis Correlative Science Studies. 41 had high OCT-1 activity (>7.2ng/200,000 cells) as defined in our original study. Patients with high OCT-1 activity had a markedly superior rate of MMR, on either standard or high dose imatinib (table 1). Significantly, a greater proportion of patients with low OCT-1 activity achieved MMR on the high dose arm compared to those on standard dose. This finding was not evident in the high OCT-1 activity group.The % of patients achieving MMR by 12 months (n)TotalLow OCT-1 ActivityHigh OCT-1 Activityp- valueTotal48% (90)90% (41)<0.001400mg59% (34)24% (17)94% (17)<0.001800mg62% (97)53% (73)87% (24)0.044p-value0.270.0120.64Table 1: The % of patients achieving MMR based on OCT-1 activity and Randomised dose.The median OCT-1 activity for those patients achieving a MMR (n=80) was 6.05ng/200,000 cells compared to 3.9 for those patients failing to achieve MMR (n=51:p=0.003). Of the 131 patients, trough imatinib levels were available on 61. A greater proportion of patients with a trough imatinib plasma level of >1000ng/ml 3 (n=50) at 1 month achieved MMR (88%) compared to those with plasma levels of <1000ng/ml (n=11:45%: p=0.032). Importantly, OCT-1 activity is not significantly different comparing those patients with trough levels >1000ng/ml (5.6ng/200,000 cells) at 1 month to those with lower trough levels (7.3ng/200,000 cells: p=0.117). This indicates that OCT-1 activity is not providing a surrogate marker of imatinib PK. Dividing the imatinib PK data into quartiles, there is no significant difference in the % of patients achieving MMR based on trough imatinib levels, in patients with high OCT-1 activity. In contrast significantly fewer patients with low OCT-1 activity and low trough levels achieve MMR by 12 months. (Table 2)The % of patients achieving MMR by 12 months (n)Imatinib PK (ng/ml)TotalLow OCT-1 ActivityHigh OCT-1 ActivityP valueQuartile 1 <160047% (15)12% (8)86% (7)0.013Quartile 2 >1600 <250080% (15)67% (6)89% (9)0.469Quartile 3 >2500 <350080% (15)77% (13)100% (2)0.654Quartile 4 >350075% (16)60% (10)91% (6)0.559Table 2: The percentage of patients achieving MMR based on quartile analysis of imatinib PK at day 29In the Australian cohort of 60 patients where detailed molecular response data is available the median molecular response in the 4 subgroups at 12 months shows a significant difference between the 400 mg group with low and high OCT-1 activity (Median BCR-ABL 0.2% IS v 0.02% IS p=0.03) but no difference in the 800 mg groups (low OCT-1 activity v high Median BCR-ABL 0.05% IS v 0.03% IS p=0.139). These analyses support our original proposal that OCT-1 activity defined at diagnosis has a major impact on molecular response and raises the possibility of patient-specific dosing. Patients with low OCT-1 activity are likely to achieve superior molecular responses if they receive imatinib at doses greater than 400 mg, whereas we could not find evidence of a molecular benefit to high dose imatinib for patients with high OCT-1 activity. The clinical value of monitoring trough imatinib drug levels remains to be clearly defined but it is likely to be greatly enhanced if it assessed in the context of the patient's OCT-1 activity.

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

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

Pharmacogenomic Factors Such as the Expression of Imatinib Transporters (OCT-1, ABCB-1 and ABCG-2) at Diagnosis, OCT-1 SNPs, and Steady-State Imatinib and Desmethyl-Imatinib Trough Plasma Levels in De Novo Chronic Phase Chronic Myelogenous Leukemia, May Influence Disease Response to Imatinib

The treatment of chronic myelogenous leukemia (CML) with imatinib mesylate (IM) has dramatically improved the prognosis of this disease, notably for chronic phase (CP) patients. However, a significant proportion of CP patients may progress along the years and it is important to identify early markers of poor response in order to offer to such patients the best alternative treatment. It has been demonstrated recently that the activity of some membrane transporters such as the organic cation transporter 1 (OCT-1), and ATP-binding cassette transporters (ABCB-1 and ABCG-2) may modify IM intracellular concentrations within leukemic cells and influence disease response and survival. In this study, we enrolled 76 CP CML patients (47M, 29F) at diagnosis and we measured in 66 patients on peripheral blood (PB) samples with RQ-PCR, the levels of OCT-1, ABCB-1 and ABCG-2 and analyse the impact on disease response and steady-state plasmatic IM and desmethyl-IM trough concentrations. The transporter data generated were normalised using a pool of peripheral blood samples from 10 healthy blood donors. Additionally, OCT-1 SNPs in exons 1 to 9 have been analysed in 41 patients out of 76. Median age at diagnosis was 50.3 (18.1–81.8) years, Sokal score was high for 16 (21%), intermediate for 34 (45%) and low for 23 (30%) patients and unknown for 3 (4%), Hasford score was high for 5 (7%), intermediate for 29 (38%), low for 30 (39%), and unknown 12 (16%). IM alone was initiated at 400 mg/day for 50 patients, 600 mg/day for 3 patients, IM was associated to monthly subcutaneous courses of 14 days Cytarabine in 13 patients and to Peg-IFN-a in 10 patients the first year of treatment. Heighty eight percent of patients were in optimal cytogenetic response (PCyR) at 6 months. Seven percent, 33%, 39%, 46%, 59% and 63% of patients were in major molecular response [MMR, BCR-ABL/ABL (IS) <0.1%] at 3, 6, 9, 12, 18 and 24 months respectively. Univariate analysis demonstrated a significant correlation (p=0.04) between ABCG-2 RQ-PCR levels at diagnosis and the proportion of patients in MMR at 6 months, between ABCB-1 levels and MMR at 12 months (p<0.01), and between OCT-1 levels and MMR at 24 months. A conditional logistic-regression model was used to analyse associations between molecular responses and transporter RQ-PCR levels, age, gender, Sokal and Hasford scores, and showed that only ABCB-1 levels were a favourable factor on the MMR rate at 12 months (HR= 2.62, 95%CI= 2.32–6.10). Logrank test did not find any significant impact of transporters levels, IM and desmethyl-IM trough concentrations, on disease progression. More extensive statistical analysis will be presented. OCT-1 SNPs analysis identified 3 patients with polymporphisms reported to decrease the enzymatic activity of OCT-1 (exon 1) and 1 patient with a polymorphism reported to fully abolish OCT-1 activity (exon 1). However, these 4 patients showed optimal cytogenetic and molecular responses, suggesting the involvement of other mechanisms in the regulation of the response to IM. Ten patients were identified with OCT-1 polymorphisms (in exons 2, 3 and 7) reported not to influence the enzymatic activity of this protein. In conclusion, the activity of these transporters (as assessed by RQ-PCR) may have some impact on the molecular response of CP CML to front-line IM therapy, however, the interrelationships between the different transporters in conjunction with other factors (drug-drug interactions, genetic background…) do not contribute to clearly distinguish one transporter more than another as a key prognostic factor for IM-molecular disease control.

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.