Intrinsic sensitivity of newly diagnosed chronic myeloid leukemia (CML) patients to imatinib (IC50imatinib) correlates with molecular response. IC50imatinib is defined as the in vitro concentration of drug required to reduce phosphorylation of the adaptor protein Crkl by 50%. We now show that interpatient variability in IC50imatinib is mainly due to differences in the efficiency of imatinib intracellular uptake and retention (IUR). In 25 untreated CML patients, the IC50imatinib strongly correlated (R2 = –0.484, P = .014 at 2 μM imatinib) with the IUR of [14C]imatinib. The addition of prazosin, a potent inhibitor of OCT-1 cellular transporter, reduced the IUR and eliminated interpatient variability. IC50 values for the more potent BCR-ABL inhibitor nilotinib (AMN107) did not correlate with IC50imatinib (R2 =–0.0561, P > .05). There was also no correlation between IC50nilotinib and the IUR for [14C]nilotinib (R2 = 0.457, P > .05). Prazosin had no effect on nilotinib IUR, suggesting that influx of nilotinib is not mediated by OCT-1. In conclusion, whereas OCT-1–mediated influx may be a key determinant of molecular response to imatinib, it is unlikely to impact on cellular uptake and patient response to nilotinib. Determining interpatient and interdrug differences in cellular uptake and retention could allow individual optimization of kinase inhibitor therapy.
Interpatient 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.
Purpose:The organic cation transporter OCT-1mediates active transport of imatinib.We recently showed that low OCT-1activity is a major contributor to suboptimal response in chronic myeloid leukemia (CML) patients treated with imatinib. The relevance of OCT-1activity and efflux pumps in determining intracellular uptake and retention (IUR) of dasatinib was assessed. Experimental Design: The effect of OCT inhibitors on [ 14 C]dasatinib and [ 14 C]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. Results: Unlike imatinib, there was no significant difference in the dasatinib IUR at 37jC and 4jC (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 IC 50 dasatinib values equivalent to patients with low OCT-1acti-vity. Dasatinib IUR was significantly lower in ABCB1-overexpressing cell lines compared with parental cell lines (P < 0.05). PSC833 (ABCB1inhibitor) significantly increased the dasatinib IUR (P < 0.05) and reduced IC 50 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 . Conclusion: 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, ABCB1and ABCG2.
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.
Most patients with de novo chronic myeloid leukemia (CML) achieve good responses to imatinib, but the rate and degree of molecular response is variable. We assessed the inhibitory concentration 50% for imatinib (IC50 imatinib ) in 62 patients with de novo chronic-phase CML as a predictor of molecular response. IC50 imatinib was determined in pretherapy blood samples by measuring the in vitro imatinib-induced reduction of the phosphorylated form of the adaptor protein Crkl (CT10 regulator of kinase like). There was marked variability between patients, with IC50 imatinib ranging from 0.375 to 1.8 M (median, 0.6 M). Patients with low IC50 imatinib (IC50 < 0.6 M; n ؍ 36) had a 36% probability of achieving 2-log reduction in BCR-ABL (breakpoint cluster region-abelson) by 3 months compared with 8% in patients with high IC50 imatinib (n ؍ 26) (P ؍ .01). The IC50 imatinib was also predictive of molecular response at 12 months, with 47% of patients in the low IC50 imatinib group achieving 3-log reduction and 23% in the high IC50 imatinib group (P ؍ .03). The predictive power of IC50 imatinib was particularly strong in patients with low Sokal scores. These data provide strong evidence that intrinsic sensitivity to imatinib is variable in previously untreated patients with CML, and the actual level of BCR-ABL kinase inhibition achieved is critical to imatinib response. The IntroductionChronic myeloid leukemia (CML) is a clonal hematologic malignancy characterized by the presence of the Philadelphia chromosome (Ph) which arises from a balanced translocation between chromosomes 9 and 22. This translocation results in the fusion of the ABL gene on chromosome 9 with the BCR gene on chromosome 22. [1][2][3] The resulting BCR-ABL (breakpoint cluster regionabelson) fusion protein is a constitutively active tyrosine kinase, conferring enhanced proliferative activity and decreased sensitivity to apoptotic cell death in the cells in which it is expressed. As such, BCR-ABL is critical to the pathogenesis of the disease. [4][5][6] Imatinib (Glivec, formerly STI571; Novartis Pharmaceuticals) is a 2-phenylaminopyramidine compound that is a potent inhibitor of all ABL tyrosine kinases (c-ABL, BCR-ABL, and Tel/ABL). In addition, imatinib is an inhibitor of the tyrosine kinases of the platelet-derived growth factor (PDGF) receptor, 7,8 ARG 9 and c-Kit, 7,10 and the macrophage colony-stimulating receptor, c-fms. 11 Clinical trials have demonstrated the efficacy of imatinib in chronic-phase CML. 12,13 Approximately 95% of patients with de novo chronic-phase CML achieve complete hematologic response, with 90% achieving major, and 80% complete, cytogenetic remissions. 14 At the molecular level, significant reductions in the level of BCR-ABL transcript as measured by quantitative real-time PCR (RQ-PCR) have been observed. 15 Although these outcomes demonstrate a significant role for imatinib in the treatment of CML, the response of some patients remains suboptimal.In the International Randomized Study of Interferon versus STI571 (IRIS)...
In chronic myeloid leukemia (CML) cell lines, brief exposure to pharmacologically relevant dasatinib concentrations results in apoptosis. In this study, we assess the impact of intensity and duration of Bcr-Abl kinase inhibition on primary CD34(+) progenitors of chronic phase CML patients. As CML cells exposed to dasatinib in vivo are in a cytokine-rich environment, we also assessed the effect of cytokines (six growth factors cocktail or granulocyte-macrophage colony-stimulating factor (CSF) or granulocyte-CSF) in combination with dasatinib. In the presence of cytokines, short-term intense Bcr-Abl kinase inhibition (>or=90% p-Crkl inhibition) with 100 nM dasatinib did not reduce CD34(+) colony-forming cells (CFCs). In contrast, without cytokines, short-term exposure to dasatinib reduced CML-CD34(+) CFCs by 70-80%. When cytokines were added immediately after short-term exposure to dasatinib, CML-CD34(+) cells remained viable, suggesting that oncogene dependence of these cells can be overcome by concomitant or subsequent exposure to cytokines. Additional inhibition of Janus tyrosine kinase (Jak) activity re-established the sensitivity of CML progenitors to intense Bcr-Abl kinase inhibition despite the presence of cytokines. These findings support the contention that therapeutic strategies combining intense Bcr-Abl kinase inhibition and blockade of cytokine signaling pathways can be effective for eradication of CML progenitors.
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