Generation of Resistance Cell Lines to AMN107, a New Inhibitor of BCR-ABL and Its Effects on Cell Lines Sensitive and Resistant to Imatinib.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4670-4670
Author(s):  
Francois-xavier Mahon ◽  
Valerie Lagarde ◽  
Paul W. Manley ◽  
Jean-Max Pasquet ◽  
Beatrice Turcq ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Resistant Cell ◽  
Myelogenous Leukemia ◽  
Cross Resistance ◽  
Main Concern ◽  
Imatinib Resistance ◽  
Abl Tyrosine Kinase ◽  
Imatinib Resistant ◽  
Clinical Resistance

Abstract Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in Chronic Myelogenous Leukemia (CML) and in Bcr-Abl positive Acute Lymphoblastic Leukemia. Imatinib is a selective inhibitor of Bcr-Abl tyrosine kinase and is now used in frontline therapy for CML. However clinical resistance is the main concern using this treatment, mediated by mutations within the kinase domain of Bcr-Abl, amplification of the BCR-ABL genomic locus or other as yet unknown mechanisms. AMN107 (Novartis Pharma AG, Basel, Switzerland) is a synthetic, second generation inhibitor of Bcr-Abl tyrosine kinase. In the current study, we tested AMN107 against different Bcr-Abl positive cell lines such as K562, LAMA84, AR230 or murine Ba/F3 cells transfected with BCR-ABL (BaF/BCR-ABL). In a 4 day proliferation assay (MTS) the dose of AMN107 that inhibited 90% of the cells (IC90) was 0.01 μM which was 100-fold lower than the IC90 of imatinib. In addition, proliferation of imatinib resistant cell lines which exhibited amplification of BCR-ABL was inhibited by 75% in the presence of 0.01 μM AMN107. However, Ba/F3 cells expressing the imatinib resistant BCR-ABL T315I mutant were only inhibited with 10μM of AMN107. Furthermore, K562-R, an imatinib-resistant cell line exhibiting a new mechanism of imatinib resistance (modification of chaperone proteins such as heat shock proteins) was also insensitive to AMN107; the IC90 for AMN107 at day 4 was 1μM versus 4μM for imatinib. Finally, we investigated potential resistance to AMN107 in Bcr-Abl positive cells. Resistant cell lines were generated after long-term (2 month) gradual dose-escalation exposure to the inhibitor. Up to now, we have obtained four cell lines, AR230-ra, K562-ra, LAMA 84-ra, and BaF/BCR-ABL-ra resistant to 8, 10, 10, and 100 nM of AMN107, respectively. Resistance was defined as the capacity to survive in the continuous presence of doses of AMN107 that kill in three days more than 90% of the parental cells in liquid culture. Preliminary investigations of AMN107 resistance using western blot and cytometry have shown that only BaF/BCR-ABL-ra overexpressed wildtype Bcr-Abl as we have already reported for imatinib resistance. Studies with these resistant cell lines investigating cross resistance with imatinib and looking for additional mechanisms of resistance are in progress.We conclude that in vitro, AMN107 is more powerful than imatinib in inhibiting the proliferation of BCR-ABL positive cell lines. In addition, we have demonstrated that it is possible to develop resistant cell lines to this new inhibitor of Bcr-Abl.

Synergy between imatinib and mycophenolic acid in inducing apoptosis in cell lines expressing Bcr-Abl

Blood ◽  
2005 ◽  
Vol 105 (8) ◽  
pp. 3270-3277 ◽  
Author(s):  
Jing Jin Gu ◽  
Lalaine Santiago ◽  
Beverly S. Mitchell
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Mycophenolic Acid ◽  
Single Agent ◽  
Myelogenous Leukemia ◽  
Molecular Response ◽  
Inosine Monophosphate Dehydrogenase ◽  
Imatinib Resistance ◽  
Abl Tyrosine Kinase ◽  
Number Of Patients

AbstractBcr-Abl tyrosine kinase activity initiates a number of intracellular signaling cascades that result in leukemogenesis. Imatinib mesylate, a specific Bcr-Abl tyrosine kinase inhibitor, has been highly successful in the treatment of chronic myelogenous leukemia (CML). However, the emergence of imatinib resistance and the incomplete molecular response of a significant number of patients receiving this therapy have led to a search for combinations of drugs that will enhance the efficacy of imatinib. We have demonstrated that mycophenolic acid (MPA), a specific inosine monophosphate dehydrogenase (IMPDH) inhibitor that results in depletion of intracellular guanine nucleotides, is synergistic with imatinib in inducing apoptosis in Bcr-Abl-expressing cell lines. Studies of signaling pathways downstream of Bcr-Abl demonstrated that the addition of MPA to imatinib reduced the phosphorylation of both Stat5 and Lyn, a Src kinase family member. The phosphorylation of S6 ribosomal protein was also greatly reduced. These results demonstrate that inhibitors of guanine nucleotide biosynthesis may synergize with imatinib in reducing the levels of minimal residual disease in CML and lay the foundation for clinical trials in which IMPDH inhibitors are added to imatinib in patients who have suboptimal molecular responses to single agent therapy or who have progressive disease. (Blood. 2005; 105:3270-3277)

In vitro efficacy of combined treatment depends on the underlying mechanism of resistance in imatinib-resistant Bcr-Abl–positive cell lines

Blood ◽  
2004 ◽  
Vol 103 (1) ◽  
pp. 208-215 ◽  
Author(s):  
Paul La Rosée ◽  
Kara Johnson ◽  
Amie S. Corbin ◽  
Eric P. Stoffregen ◽  
Erika M. Moseson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Durable Resistance ◽  
Combined Treatment ◽  
Chronic Phase ◽  
Resistant Cell ◽  
Myelogenous Leukemia ◽  
Rational Approach ◽  
Resistance Testing ◽  
Cross Resistance ◽  
Imatinib Resistant

Abstract Imatinib mesylate (Gleevec, formerly STI571) is an effective therapy for all stages of chronic myelogenous leukemia (CML). While responses in chronic-phase CML are generally durable, resistance develops in many patients with advanced disease. We evaluated novel antileukemic agents for their potential to overcome resistance in various imatinib-resistant cell lines. Using cell proliferation assays, we investigated whether different mechanisms of resistance to imatinib would alter the efficacy of arsenic trioxide (As2O3) or 5-aza-2-deoxycytidine (decitabine) alone and in combination with imatinib. Our results indicate that resistance to imatinib induced by Bcr-Abl overexpression or by engineered expression of clinically relevant Bcr-Abl mutants does not induce cross-resistance to As2O3 or decitabine. Combined treatment with these agents and imatinib is beneficial in cell lines that have residual sensitivity to imatinib monotherapy, with synergistic growth inhibition achieved only at doses of imatinib that overcome resistance. In some imatinib-resistant cell lines, combination treatments that use low doses of imatinib lead to antagonism. Apoptosis studies suggest that this can be explained in part by the reduced proapoptotic activity of imatinib in resistant cell lines. These data underline the importance of resistance testing and provide a rational approach for dose-adjusted administration of imatinib when combined with other agents.

scholarly journals Prolonged exposure to FLT3 inhibitors leads to resistance via activation of parallel signaling pathways

Blood ◽  
2006 ◽  
Vol 109 (4) ◽  
pp. 1643-1652 ◽  
Author(s):  
Obdulio Piloto ◽  
Melissa Wright ◽  
Patrick Brown ◽  
Kyu-Tae Kim ◽  
Mark Levis ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Prolonged Exposure ◽  
Cellular Transformation ◽  
Mapk Signaling ◽  
Resistant Cell ◽  
Myelogenous Leukemia ◽  
Continuous Treatment

Abstract Continuous treatment of malignancies with tyrosine kinase inhibitors (TKIs) may select for resistant clones (ie, imatinib mesylate). To study resistance to TKIs targeting FLT3, a receptor tyrosine kinase that is frequently mutated in acute myelogenous leukemia (AML), we developed resistant human cell lines through prolonged coculture with FLT3 TKIs. FLT3 TKI-resistant cell lines and primary samples still exhibit inhibition of FLT3 phosphorylation on FLT3 TKI treatment. However, FLT3 TKI-resistant cell lines and primary samples often show continued activation of downstream PI3K/Akt and/or Ras/MEK/MAPK signaling pathways as well as continued expression of genes involved in FLT3-mediated cellular transformation. Inhibition of these signaling pathways restores partial sensitivity to FLT3 TKIs. Mutational screening of FLT3 TKI-resistant cell lines revealed activating N-Ras mutations in 2 cell lines that were not present in the parental FLT3 TKI-sensitive cell line. Taken together, these data indicate that FLT3 TKI-resistant cells most frequently become FLT3 independent because of activation of parallel signaling pathways that provide compensatory survival/proliferation signals when FLT3 is inhibited. Anti-FLT3 mAb treatment was still cytotoxic to FLT3 TKI-resistant clones. An approach combining FLT3 TKIs with anti-FLT3 antibodies and/or inhibitors of important pathways downstream of FLT3 may reduce the chances of developing resistance.

Application of FASTTM Fragment-Based Lead Discovery and Structure-Guided Design to Discovery of Small Molecule Inhibitors of BCR-ABL Tyrosine Kinase Active Against the T315I Imatinib-Resistant Mutant.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 698-698 ◽  
Author(s):  
Stephen K. Burley
Keyword(s):  
Tyrosine Kinase ◽  
Fusion Gene ◽  
Clinical Success ◽  
Philadelphia Chromosome ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Lead Discovery ◽  
Abl Tyrosine Kinase ◽  
Imatinib Resistant

Abstract The Philadelphia chromosome translocation creates a BCR-ABL fusion gene that encodes a constitutively active BCR-ABL tyrosine kinase, which gives rise to chronic myelogenous leukemia (CML). The clinical success of imatinib (Gleevec) demonstrated that BCR-ABL tyrosine kinase inhibitors can provide effective treatment for CML. However, some CML patients treated with imatinib develop resistance leading to disease progression. The majority of resistance is due to point mutations in BCR-ABL, which give rise to active mutant enzymes that are insensitive to imatinib. In all, ~30 imatinib-resistant BCR-ABL mutants have been identified in clinical isolates. The T315I mutant represents ~20% of clinically observed mutations, making it one of the most common causes of resistance. Second-generation BCR-ABL inhibitors, including AMN-107 and BMS-354825, inhibit many of the clinically relevant mutants but not T315I. Mutant T315I BCR-ABL is, therefore, an important and challenging target for discovery of CML therapeutics. We have applied a proprietary X-ray crystallographic fragment-based lead discovery platform (FASTTM) and structure-guided lead optimization to identify potent inhibitors of wild-type BCR-ABL and the four most common mutants, including T315I. Our lead discovery efforts yielded five chemical series that inhibit both wild-type (WT) and T315I BCR-ABL. Compounds in our most advanced lead series potently inhibit proliferation of K562 cells and Ba/F3 cells with WT BCR-ABL and the four major clinically relevant BCR-ABL mutations (T315I, E255K, M351T, Y253F; see below). Further details describing in vitro and in vivo profiling of these novel BCR-ABL T315I inhibitors will be presented. Ba/F3 cell proliferation for BCR-ABL Inhibitors (EC50, nM) BCR-ABL Form Imatinib AMN-107 BMS-354825 SGX-70430 WT 790 33 12 11 T315I > 10000 > 10000 > 10000 21 Y253F 5700 370 8 334 E255K 8300 350 7 77 M351T 2000 38 28 15 Control Assay Ba/F3 (T315I) + IL3 > 10000 > 10000 > 10000 > 10000

Jak2: A Potential Therapeutic Target for Chronic Myelogenous Leukemia (CML).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2121-2121
Author(s):  
Ajoy K. Samanta ◽  
Hui Lin ◽  
Tong Sun ◽  
Hagop Kantarjian ◽  
Ralph B. Arlinghaus
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Leukemia Cells ◽  
Imatinib Treatment ◽  
Potential Therapeutic Target ◽  
Imatinib Resistant

Abstract In most CML patients Bcr-Abl, a constitutively active tyrosine kinase derived from the Philadelphia chromosome, is highly expressed and is the causative factor in most CML patients. Imatinib mesylate, an inhibitor of the Bcr-Abl kinase, is a very effective drug for treatment of CML. However in some CML patients, drug resistance develops and the patients relapse. Thus, alternative drug targets need to be identified. We have shown that Bcr-Abl activates its downstream target, the Jak2 tyrosine kinase, leading to the enhancement of c-Myc expression (Xie et al. Oncogene21: 7137, 2002; Samanta et al. Cancer Res.66: 6468, 2006). Our recent studies showed that Bcr-Abl activated the transcriptional factor NF-kB through Jak2, which in turn activated c-Myc transcription. Jak2 also activated Akt, which increased c-Myc protein levels by inhibiting GSK3. Addition of AG490, an inhibitor of the Jak2 kinase, prevented enhanced expression of c-Myc and caused induction of apoptosis in BCR-ABL+ leukemia cells. Immunoprecipitation experiments showed that Bcr-Abl is associated with a cluster of signaling proteins including Jak2, Gab2, Akt and GSK3b. Treatment of CML cell lines and mouse BCR-ABL+ 32D cells (myeloid lineage) with the either Jak2 siRNA or the Jak2 kinase inhibitor AG490 caused inhibition of pTyr Gab2 formation, pSer Akt formation and the activation of NFkB. Of interest, treatment of BCR-ABL+ 32 D cells with IL-3 reversed the apoptotic effects of imatinib by activation of Jak2 even though Bcr-Abl was inhibited. Importantly, mouse BaF3 hematopoietic cells expressing the T315I and E255K imatinib-resistant mutants of BCR-ABL underwent apoptosis upon exposure to either the Jak2 inhibitor AG490 or siRNA for Jak2, yet were resistant to imatinib. Cells from a number of CML patients (including six chronic phase, one accelerated phase, and two blast crisis patients who failed imatinib treatment) were induced to enter apoptosis upon treatment with AG490, whereas normal samples were not affected by AG490. Further analysis of imatinib resistant Bcr-Abl cell lines showed that transfection of the cells with Jak2 specific siRNA or by treating the cells with AG490 reduced levels of pLyn, pAkt, c-Myc and pGSK3 level compared to untreated cells. Transfection of Lyn specific siRNA into K562 and 32Dp210 cells resulted in down-regulation of pGab2, pAkt, pGsk3 and c-Myc, but did not alter pJak2 levels; this result indicates that pLyn is downstream of Jak2 but upstream of Gab2, pAkt, pGSK3 in BCR-ABL+ leukemia cells. We hypothesize that Jak2 activation of Lyn tyrosine kinase in BCR-ABL+ leukemia cells leads to tyrosine phosphorylation of the YxxM motif of Gab2, which activates the PI-3 kinase-Akt pathway. In conclusion, since inactivation of Jak2 inhibits many of the critical oncogenic targets of Bcr-Abl (resulting in apoptosis induction), we propose that Jak2 is a potential therapeutic target for CML, in both imatinib sensitive and imatinib resistant patients.

Induction of Apoptosis In Imatinib-Resistant BCR-ABL1-Positive Leukemia Cell Lines by Bortezomib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4469-4469
Author(s):  
Hilmar Quentmeier ◽  
Sonja Eberth ◽  
Julia Romani ◽  
Margarete Zaborski ◽  
Hans G. Drexler
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Leukemia Cell ◽  
Resistant Cell ◽  
Imatinib Resistance ◽  
Imatinib Resistant ◽  
Induced Apoptosis

Abstract Abstract 4469 The BCR-ABL1 translocation occurs in chronic myeloid leukemia (CML) and in 25% of cases with acute lymphoblastic leukemia (ALL). We screened a panel of BCR-ABL1 positive cell lines to find models for imatinib-resistance studies. Five of 19 BCR-ABL1 positive cell lines were resistant to imatinib-induced apoptosis (KCL-22, MHH-TALL1, NALM-1, SD-1, SUP-B15). None of the five resistant cell lines carried mutations in the kinase domain of BCR-ABL1 and – consequently – all also showed resistance to the second generation kinase inhibitors, nilotinib or dasatinib. All Philadelphia chromosome (Ph)-positive cell lines demonstrated constitutive phosphorylation of STAT5 and S6. Imatinib induced dephosphorylation of both BCR-ABL1 downstream effectors in responsive cell lines, but - remarkably – induced dephosphorylation of STAT5 in resistant cell lines as well. By administering well-described signalling pathway inhibitors we were able to show that activation of mTOR complex 1 was responsible for the constitutive S6 phosphorylation of imatinib-resistant cells. Neither BCR-ABL1 nor Src kinases or Ras/Rac-GTPases underlie tyrosine kinase inhibitor resistance in these cell lines. In conclusion, none of the five TKI-resistant cell lines showed aberrant activation of previously-described oncogenic pathways which would explain their resistance. These findings raise the question whether these cell lines might help to find a novel – alternative – explanation for TKI resistance. Interestingly, the proteasome inhibitor bortezomib induced apoptosis in TKI-resistant and –sensitive Ph+ cell lines. Bortezomib is being used for the treatment of multiple myeloma. Our findings support the notion that bortezomib might also be useful for the treatment of imatinib-resistant CML. Disclosures: No relevant conflicts of interest to declare.

Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, is effective in patients with Philadelphia chromosome–positive chronic myelogenous leukemia in chronic phase following imatinib resistance and intolerance

Blood ◽  
2007 ◽  
Vol 110 (10) ◽  
pp. 3540-3546 ◽  
Author(s):  
Hagop M. Kantarjian ◽  
Francis Giles ◽  
Norbert Gattermann ◽  
Kapil Bhalla ◽  
Giuliana Alimena ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Kinase Inhibitor ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Imatinib Resistance ◽  
Abl Tyrosine Kinase ◽  
Abl Tyrosine Kinase Inhibitor ◽  
Philadelphia Chromosome Positive

Abstract Nilotinib, an orally bioavailable, selective Bcr-Abl tyrosine kinase inhibitor, is 30-fold more potent than imatinib in pre-clinical models, and overcomes most imatinib resistant BCR-ABL mutations. In this phase 2 open-label study, 400 mg nilotinib was administered orally twice daily to 280 patients with Philadelphia chromosome–positive (Ph+) chronic myeloid leukemia in chronic phase (CML-CP) after imatinib failure or intolerance. Patients had at least 6 months of follow-up and were evaluated for hematologic and cytogenetic responses, as well as for safety and overall survival. At 6 months, the rate of major cytogenetic response (Ph ≤ 35%) was 48%: complete (Ph = 0%) in 31%, and partial (Ph = 1%-35%) in 16%. The estimated survival at 12 months was 95%. Nilotinib was effective in patients harboring BCR-ABL mutations associated with imatinib resistance (except T315I), and also in patients with a resistance mechanism independent of BCR-ABL mutations. Adverse events were mostly mild to moderate, and there was minimal cross-intolerance with imatinib. Grades 3 to 4 neutropenia and thrombocytopenia were observed in 29% of patients; pleural or pericardial effusions were observed in 1% (none were severe). In summary, nilotinib is highly active and safe in patients with CML-CP after imatinib failure or intolerance. This clinical trial is registered at http://clinicaltrials.gov as ID no. NCT00109707.

scholarly journals Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, is active in patients with imatinib-resistant or -intolerant accelerated-phase chronic myelogenous leukemia

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 1834-1839 ◽  
Author(s):  
Philipp le Coutre ◽  
Oliver G. Ottmann ◽  
Francis Giles ◽  
Dong-Wook Kim ◽  
Jorge Cortes ◽  
...  
Keyword(s):  
Adverse Events ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Chronic Myelogenous Leukemia ◽  
Kinase Inhibitor ◽  
Myelogenous Leukemia ◽  
Abl Tyrosine Kinase ◽  
Imatinib Resistant ◽  
Abl Tyrosine Kinase Inhibitor ◽  
Grade 3

Patients with imatinib-resistant or -intolerant accelerated-phase chronic myelogenous leukemia (CML-AP) have very limited therapeutic options. Nilotinib is a highly selective BCR-ABL tyrosine kinase inhibitor. This phase 2 trial was designed to characterize the efficacy and safety of nilotinib (400 mg twice daily) in this patient population with hematologic response (HR) as primary efficacy endpoint. A total of 119 patients were enrolled and had a median duration of treatment of 202 days (range, 2–611 days). An HR was observed in 56 patients (47%; 95% confidence interval [CI], 38%-56%). Major cytogenetic response (MCyR) was observed in 35 patients (29%; 95% CI, 21%-39%). The median duration of HR has not been reached. Overall survival rate among the 119 patients after 12 months of follow-up was 79% (95% CI, 70%-87%). Nonhematologic adverse events were mostly mild to moderate. Severe peripheral edema and pleural effusions were not observed. The most common grade 3 or higher hematologic adverse events were thrombocytopenia (35%) and neutropenia (21%). Grade 3 or higher bilirubin and lipase elevations occurred in 9% and 18% of patients, respectively, resulting in treatment discontinuation in one patient. In conclusion, nilotinib is an effective and well-tolerated treatment in imatinib-resistant and -intolerant CML-AP. This trial is registered at www.clinicaltrials.gov as NCT00384228.

Response dynamics to nilotinib depend on the type of BCR-ABL mutations in patients with chronic myelogenous leukemia (CML) after imatinib failure

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 7024-7024 ◽  
Author(s):  
M. C. Mueller ◽  
S. Branford ◽  
J. Radich ◽  
D. W. Kim ◽  
G. Martinelli ◽  
...  
Keyword(s):  
Amino Acids ◽  
Tyrosine Kinase ◽  
Mutant Cell ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Tyrosine Kinase Domain ◽  
Response Dynamics ◽  
Imatinib Resistance ◽  
Hematologic Response ◽  
Abl Tyrosine Kinase

7024 Background: Nilotinib (AMN107) is an oral, aminopyrimidine-derivative, selective inhibitor of the BCR-ABL tyrosine kinase with improved potency and specificity compared with imatinib. In preclinical models, activity of nilotinib was also demonstrated in 32/33 imatinib- resistant mutant cell lines. We sought to explore the efficacy of nilotinib in vivo according to the type of preexisting BCR-ABL mutations associated with imatinib resistance. Methods: We have investigated peripheral blood samples from 101 chronic phase (CP) and 41 accelerated phase (AP) CML patients (pts) who had been enrolled in a phase II study investigating the efficacy and safety of 400mg nilotinib bid after imatinib failure. Screening for BCR-ABL mutations was performed by D-HPLC combined with DNA sequencing. The analysis covered amino acids 207–517 of the BCR-ABL tyrosine kinase domain. Results: Prior to nilotinib, 24 different BCR-ABL mutations involving 20 amino acids were detected affecting 44% CP and 61% AP pts. After 6 mo of therapy, complete hematologic response was achieved in 59%, major cytogenetic response (MCR) in 25% being complete (CCR) in 16% of pts with mutations vs 81%, 51% and 33% of pts without mutations, respectively. Response dynamics were associated with preclinical activity of nilotinib: MCR was reached in 10/18 pts with mutations associated with preclinical IC50 to nilotinib of <100nM, 2/9 pts with IC50 of 100–1,000 nM, and 0/4 pts with mutation T315I demonstrating virtual resistance to imatinib and nilotinib. In AP pts, hematologic response was achieved in 56% and 31%, MCR in 24% vs 19%, and CCR in 24% vs 13% of pts with or without mutations, respectively. Conclusions: Nilotinib is efficacious in pts with BCR-ABL mutations, except T315I, as well as in patients with BCR-ABL-independent resistance. Time to response may depend on the individual type of the mutation and correlates with the IC50 to nilotinib. Thus, nilotinib may have an important therapeutic role in imatinib resistance as well as in frontline CML therapy to prevent emergence of resistant clones. [Table: see text]

The IKK Inhibitor PS1145 Allows to Overcome Imatinib Resistance.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2940-2940
Author(s):  
Daniela Cilloni ◽  
Francesca Messa ◽  
Francesca Arruga ◽  
Ilaria Defilippi ◽  
Alessandro Morotti ◽  
...  
Keyword(s):  
Cell Lines ◽  
Phase 1 ◽  
Chronic Phase ◽  
Colony Growth ◽  
Resistant Cell ◽  
Proliferation Rate ◽  
Imatinib Resistance ◽  
Inhibition Of Proliferation ◽  
Imatinib Resistant ◽  
Resistant Cells

Abstract Since a number of CML patients are resistant to Imatinib, additional molecular defects should be identified and targeted to improve the therapeutic strategies. A constitutive NF-kB activity has been demonstrated in several hematologic malignancies, therefore NF-kB blocking approaches have been introduced as antineoplastic strategies. The phosphorilation of IKB by IKK leads to IKB degradation by the proteasome, so freeing NF-kB to enter the nucleus and activate transcription. IKK may therefore represent an attractive target for molecular therapies. The aim of the study was to evaluate the effects of the IKK inhibitor PS1145 (Millenium) in CML cell lines and patients sensitive and resistant to Imatinib. K562 and KCl cells both sensitive (s) and resistant (r) to Imatinib and the BM cells collected from 13 CML patients were incubated with PS1145 10 μM, with Imatinib 1 μM and with the combination of the two drugs for 24 and 48 hrs. 11 out of 13 patients were in chronic phase, 1 in accelerate phase and 1 in blastic phase. 3 out of 11 were cytogenetic resistant, one was also hematologically resistant. The NF-kB activity was evaluated by ELISA method, the proliferation rate by MTT assay, apoptosis by FACS. Immunofluorescence was use to identify the NF-kB localization. Moreover colony growth was evaluated in control and treated samples. PS1145 was able to reduce the DNA binding activity of NF-kB of 90% and 85% respect to the control in K562 and KCL cells.This was confirmed by the prevailing cytoplasmatic localization of NF-kB after PS1145 incubation. In K562s and KCls treated with Imatinib, we observed a reduction of proliferation of 48% and 37% respectively. By contrast no effect on proliferation was observed in K562r and KClr. The incubation with PS1145 inhibited the proliferation of 38% and 15% in K562s and KCls, and of 22% and 28% in K562r and KClr. Interestingly in resistant cell lines the association of Imatinib and PS1145 induced a marked inhibition of proliferation of 87% in K562r and 82% in KClr. Similarly, PS1145 inhibited BM cells proliferation of 30% (range 11% to 65%). Imatinib incubation of BM cells from sensitive patients reduced the proliferation rate of 41% (range 29%–55%) but no effects were observed in resistant patients. In the three resistant patients the incubation with both compounds resulted in an increased block of proliferation respect to PS1145 alone with an inhibition of 42%, 49% and 58% after 24 hrs and 70%, 77% and 79% after 48 hrs. Imatinib plus PS1145 induced a significant increase of apoptosis from 7% to 69% in K562r and from 9 % to 71% in KClr. In agreement this association induced 48%, 52% and 39% of apoptotic cells in BM from the three resistant patients and a colony growth inhibition of 86%. Our data clearly demonstrated that the IKK inhibitor PS1145 is able to induce growth arrest and apoptosis in CML cell lines and BM cells. This effect is more sound in Imatinib resistant cells treated with the association of Imatinib and PS1145. Although the exact mechanism of action of PS1145 in resistant cells is at present a pure speculation, these data may suggest an intriguing mechanism to induce apoptosis in imatinib resistant cells based on imatinib resistance itself. The combination of Imatinib and the IKK inhibitor could therefore represent a valid approach for the treatment of CML patients resistant to Imatinib therapy.

Sign in / Sign up

Export Citation Format

Share Document