Imatinib Mesylate Resistance Research Articles

Low Expression of hOCT1 May Be a Key Point Mediating Low Intracellular Imatinib Accumulation in Imatinib Mesylate Resistance K562 Cell Line.

Objective To establish an imatinib resistance cell line and to study its resistant principia.Methods1.K562 cells were cultured in imatinib at gradually increased concentrations to generate their resistance cell line.2.Clone imatinib resistance cell lines by limited dilution culture.3.MTT assay, real time PCR and Semi-quantity PCR, flow cytometry and HPLC were used to clarify the possible mechanisms of the resistance.Results1.Imatinib resistance cell line K562R was successfully induced by continuous culture in the presence of gradually increasing doses of imatinib up to 5μmol/L. K562R cells were maintained in the media containing 5μmol/L imatinib.2.Proliferation data showed that cell growth of K562R was not inhibited in 5 μmol/L imatinib, whereas the parental sensitive cell was significantly inhibited by up to 2μM imatinib.3.The IC50 of K562R was about 7.5μmol/L which was ten times higher than that of the parental cell.4.HPLC revealed that the intracellular imatinib concentration of K562R was strikingly lower than that of the parental cells (up to 27.8-fold).MDR1 were not detected in mRNA (by RT-PCR)and protein(by flow cytometry) levels on K562R cell, whereas hOCT1 level measured by semi-quantity PCR showed lower expression in K562R cell lines than that of parental sensitive cell, indicating that low intracellular imatinib concentration may be due to lower affluence of imatinib by low level of hOCT1. (5) Real time PCR analysis showed no BCR-ABL/G6PD gene amplification and sequence analysis of the 374bp ABL kinase domain showed no mutation in K562R cell lines.Conclusion An imatinib resistance cell line K562R has been successfully established. Low expression of hOCT1 may be a key point mediating low intracellular imaitnib accumulation in K562R cell lines.

Modulation of P-glycoprotein and proinflammatory cytokines with HMG-CoA reductase inhibitor simvastatin (SV) in chronic myelogenous leukemia (CML) patients with imatinib mesylate (IM) resistance

17528 Background: Primarily monocyte-derived cytokines and soluble interleukin-2 receptor (sIL-2R) form a group of proinflammatory cytokines and mononuclear cell activaton respectively which are significantly elevated in patients with CML-chronic phase (CML-CP) or CML-blastic crisis (CML-BC).C-reactive protein (CRP) correlates with IL-6. IM is a tyrosine kinase inhibitor that specifically acts intracellularly by blocking the ATP-binding site of the kinase domain of Bcr/abl in CML. Acquired drug resistance in CML-CP patients treated with IM may occur by upregulation of P-glycoprotein (P-gp) drug efflux with P-gp+ leukemic cells and P-gp dependent decline of intracellular IM levels with retained phosphorylation Bcr/abl pattern and loss of IM effect on apoptosis and cellular proliferation. Modulation of P-gp with HMG-CoA reductase inhibitor (SV), a potent inhibitor of P-gp transport, readily overcame IM resistance. Methods: We present two cases of CML: CML-CP and CML-BC with evidence of IM resistance. After one year of IM therapy an 86 yo female with CML-CP stage II, developed reactivation of Bcr/abl signaling due to excretion of IM from the cell transmembrane transporters consistent with increased proinflammatory cytokines, sIL-2R, CRP, acute coronary syndrome but normal neopterin.Results: Modulation of proinflammatory cytokines with SV, IM dose adjustments, serial measurements of Bcr-abl/abl ratio didnot exceed 0.02% on three occasions or 0.05% on two occasions after changing from 0.001% to 0.003% and now after two years of IM plus SV, real-time Bcr-abl/abl ratio is 0.000 (a 5 log reduction).sIL-2R levels normalized from 31,709.55 pg/ml to 5,345.4 pg/ml (Normal 1770–9753 pg/ml). CRP decreased to 6.23 mg/dl from 16 mg/dl. Conclusions: IM an intracellular drug demonstrating high activity against Bcr/abl gene positive CML-CP or CML-BC patients may develop resistance and loss of cellular proliferation via upregulated P-gp-mediated drug efflux mechanism. Modulation of P-gp by HMG-CoA reductase (statins) inhibitor simvastatin, a potent inhibitor of P-gp, may allow intracellular levels of IM to restore its cytotoxicity and overcome resistance mechanism. No significant financial relationships to disclose.

Mechanisms of sunitinib malate (SU) resistance in gastrointestinal stromal tumors (GISTs)

10006 Background: SU, an oral, multitargeted inhibitor of KIT, PDGFRs, VEGFRs, FLT3, and RET receptor tyrosine kinases, is now the standard therapy for treatment of imatinib mesylate (IM)-resistant GIST tumors or IM-intolerant GIST patients. However, it is unclear if the clinical efficacy of SU in this setting is due to inhibition of KIT and/or PDGFRA in tumor cells, inhibition of VEGFRs and PDGFRs in endothelial cells and pericytes, respectively, or a combination of antitumor and antiangiogenic effects. In order to address this question, we studied the molecular mechanisms of SU resistance in vitro and in patient-derived tumors. Methods: We studied the in-vitro effects of IM or SU on cells with KIT exon 11 mutations, either alone or in combination with known IM-resistant secondary mutations. In addition, we analyzed tumor samples obtained from patients undergoing salvage surgery after SU treatment failure. Tumor specimens were genotyped for primary and secondary mutations. Results: SU was approximately 2–5-fold more potent than IM against typical GIST-associated KIT exon 11 mutations. In addition, the presence of secondary mutations involving the ATP/drug binding pocket (V654A or T670I) did not substantially alter SU potency. In contrast, these ATP/drug-binding-pocket mutations conferred high-level resistance to IM. However, SU had no significant inhibitory effect on the kinase activity of secondary IM-resistant mutations involving the KIT activation loop (exon 17, codons 816, 820, 822, and 823). Surgical resection was performed on two GIST patients who had clinical progression on SU. Among nine progressing lesions, substitutions were detected in codons 816, 820, and 822; a novel mutation, L783V, was also identified. In contrast, two non-progressing tumors both contained a V654A secondary mutation. Conclusions: Consistent with in-vitro studies, acquired IM-resistant mutations of the KIT activation loop in GIST are associated with clinical resistance to SU and IM. In addition, novel kinase mutations not previously associated with IM resistance may contribute to clinical SU resistance. These findings suggest that the antiangiogenic effects of SU may be insufficient to inhibit GIST progression when the targeted oncogenic kinase remains active. [Table: see text]

To assess the response and toxicity of imatinib mesylate and low-dose cytarabine in imatinib non-responder chronic myeloid leukemia, chronic phase (CML-CP) patients: A pilot study

17507 Background: To overcome primary imatinib mesylate (Gleevec ) resistance, we used a combination of imatinib mesylate and low dose cytarabine to treat patients of CML-CP. Methods: Between January 2005 and July 2006, 30 patients (median age 34 years, range 20 to 57 years, M:F ratio 5:1) were enrolled. 2/30 patients had primary hematological and 28/30 had primary cytogenetic resistance. 7 patients had been pretreated with interferon alpha. Treatment consisted of imatinib 400mg daily and cytarabine 10mg subcutaneous daily for 10 days every month. Median of 5 cycles (range 3–6) was administered. Patients were monitored on outpatient basis with weekly blood counts. Bone marrow cytogenetics was done 3 monthly to assess cytogenetic response (CGR). Toxicity was recorded as per NCI CTC (version 2). Primary end points were response and toxicity at the end of 6 cycles. We measured plasma imatinib levels by HPLC in 20 of these patients (J Chromatography B 2004;84:431–34) and compared the levels with 20 patients who were imatinib responders. Results: 11/30 (37%) patients obtained major CGR including complete CGR in 6/30 (20%). None of the patients with primary hematological resistance responded. Grade 3/4 hematological and non hematological toxicities were seen in 3.33% (n=1) and6.66% (n=2) patients respectively. 8/30 (27%) patients discontinued treatment due to reversible cardiac arrest (n=1), hepatitis (n=1), progression to BC/AP (n=2), grade 4 thrombocytopenia (n=1), loss of CHR (n=2), and consent withdrawl (n=1). The mean plasma imatinib levels in non responders were significantly lower, compared to imatinib responders (0.70 micro moles vs 2.34 micro moles, p=0.002). Conclusions: Present study confirms the activity of low dose cytarabine and imatinib in patients having primary cytogenetic resistance. The reasons for low plasma imatinib levels among non responders and methods to circumvent so as to achieve higher plasma levels of imatinib needs further study. No significant financial relationships to disclose.

Identification of BCR-ABL point mutations conferring resistance to the Abl kinase inhibitor AMN107 (nilotinib) by a random mutagenesis study

AbstractPatients with advanced stages of chronic myeloid leukemia (CML) often manifest imatinib mesylate resistance associated with point mutations in BCR-ABL. AMN107 is a new higher-potency inhibitor of BCR-ABL. To identify mutations in BCR-ABL that could result in resistance to AMN107, a cDNA library of BCR-ABL mutants was introduced into Ba/F3 cells followed by selection in AMN107 (0.125-0.5 µM). A total of 86 individual, drug-resistant colonies were recovered, and the SH3, SH2, and kinase domains of BCR-ABL were sequenced. A total of 46 colonies had single point mutations in BCR-ABL, with a total of 17 different mutations, all within the kinase domain. The other 40 colonies had multiple point mutations and were not analyzed further. Each of the 17 single point mutants were reconstructed by site-directed mutagenesis of native BCR-ABL and found to be approximately 2.5- to 800-fold more resistant to AMN107 than native BCR-ABL. The mutations included 6 known imatinib mesylate–resistant mutations, including T315I, which showed complete resistance to AMN107. Interestingly, most AMN107-resistant mutants were also resistant to imatinib mesylate. These results may predict some of the resistance mutations that will be detected in clinical trials with this kinase inhibitor.

Second-generation tyrosine kinase inhibitors as therapy for chronic myeloid leukemia

Chronic myeloid leukemia (CML) was the first human malignancy to be associated with a single genetic abnormality, characterized by a reciprocal translocation involving chromosomes 9 and 22 (the Philadelphia chromosome). The fusion gene that results (BCR-ABL) produces a constitutively activated tyrosine kinase that exists in different isoforms depending on BCR break-points. Imatinib mesylate is a highly selective inhibitor of this kinase, producing normal blood-counts in 98% of patients in chronic phase CML and disappearance of the Philadelphia chromosome in 86%. However, 17% of patients in the chronic phase will either relapse or develop resistance resulting mainly from one or more point mutations affecting at least 30 amino acids within the Abl kinase protein. This review focuses on the relevant biology of CML, imatinib mesylate resistance mechanisms, and the current status of the next generation of Bcr-Abl inhibitors.

MEK1/2 inhibitors sensitize Bcr/Abl+ human leukemia cells to the dual Abl/Src inhibitor BMS-354/825

AbstractInteractions between MEK1/2 inhibitors and the dual Abl/Src kinase inhibitor dasatinib (BMS-354825) were examined in chronic myeloid leukemia (CML) cell lines and primary specimens. Cotreatment of K562 or LAMA cells with subtoxic or marginally toxic concentrations of PD184352 (or U0126) and dasatinib synergistically potentiated mitochondrial damage, caspase activation, and apoptosis. Similar interactions were observed in CD34+ cells from one CML patient–derived but not in a normal human CD34+ bone marrow cell specimen. These interactions were associated with multiple perturbations in survival signaling pathways, including inactivation of Bcr/Abl, STAT5, and ERK1/2; down-regulation of Bcl-xL and Mcl-1; and dephosphorylation/activation of Bim. They were also associated with BAX/BAK conformational change, mitochondrial dysfunction, and caspase activation. Bim knockdown by shRNA suppressed BAX and BAK conformational change and protected cells from dasatinib/PD184352 lethality. Conversely, K562 cells ectopically expressing Mcl-1 or Bcl-xL were significantly less susceptible to dasatinib/PD184352 toxicity. Notably, the dasatinib/PD184352 regimen was active against leukemic cells exhibiting various forms of imatinib mesylate resistance, including Bcr/Abl overexpression, Lyn activation, and several Bcr/Abl kinase domain mutations (eg, E255K, M351T), but not T315I. Together, these findings suggest that strategies combining dasatanib with MEK1/2 inhibitors warrant further investigation in Bcr/Abl+ malignancies, particularly in the setting of imatinib mesylate–resistant disease.

A Review of Resistance Patterns and Phenotypic Changes in Gastrointestinal Stromal Tumors Following Imatinib Mesylate Therapy

Gastrointestinal stromal tumors are neoplastic lesions that arise from the interstitial cells of Cajal and are associated with somatic mutations in the tyrosine kinase receptor, KIT. The only known curative therapy is complete surgical resection. Unfortunately, postsurgical recurrence rates exceed 50% and most tumors are resistant to standard chemotherapy and radiation. Imatinib mesylate, a novel tyrosine kinase inhibitor, holds promise as a potential adjuvant therapy to prevent recurrence and improve long-term survival. However, as resistance data emerge, it appears that a potential “escape pathway” may originate from secondary mutations in the KIT receptor. This paper reviews the historical clinical experience with imatinib mesylate and discusses resistance patterns following targeted therapy. We highlight this review with an interesting case report that illustrates unique phenotypic tumoral changes associated with imatinib mesylate resistance.

Adaptive secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates imatinib and nilotinib resistance in BCR/ABL+ progenitors via JAK-2/STAT-5 pathway activation

AbstractOvercoming imatinib mesylate (IM) resistance and disease persistence in patients with chronic myeloid leukemia (CML) is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL+ cells in the presence of IM and nilotinib (NI; AMN107), a novel, more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84 cell clones (R-CM) was found to substantially protect IM-naive LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony-stimulating factor (GM-CSF), which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the janus kinases 2 (JAK-2)/signal transducer and activator of transcription 5 (STAT-5) signaling pathway in GM-CSF receptor α receptor (CD116)–expressing cells, including primary CD34+/CD116+ GM progenitors (GMPs). Elevated mRNA and protein levels of GM-CSF were detected in IM-resistant patient samples, suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly, inhibition of JAK-2 with AG490 abrogated GM-CSF–mediated STAT-5 phosphorylation and NI resistance in vitro. Together, adaptive autocrine secretion of GM-CSF mediates BCR/ABL-independent IM and NI resistance via activation of the antiapoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF–induced IM and NI progenitor cell resistance, providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML.

Chronic myeloid leukemia stem cells possess multiple unique features of resistance to BCR-ABL targeted therapies

The leukemic stem cells in patients with chronic myeloid leukemia (CML) are well known to be clinically resistant to conventional chemotherapy and may also be relatively resistant to BCR-ABL-targeted drugs. Here we show that the lesser effect of imatinib mesylate (IM) on the 3-week output of cells produced in vitro from lin(-)CD34(+)CD38(-) CML (stem) cells compared with cultures initiated with the CD38(+) subset of lin(-)CD34(+) cells is markedly enhanced (>10-fold) when conditions of reduced growth factor stimulation are used. Quantitative analysis of genes expressed in these different CML subsets revealed a differentiation-associated decrease in IL-3 and G-CSF transcripts, a much more profound decrease in expression of BCR-ABL than predicted by changes in BCR expression, decreasing expression of ABCB1/MDR and ABCG2 and increasing expression of OCT1. p210(BCR-ABL) and kinase activity were also higher in the lin(-)CD34(+)CD38(-) cells and formal evidence that increasing BCR-ABL expression decreases IM sensitivity was obtained from experiments with a cell line model. Nevertheless, within the entire CD34(+) subset of CML cells, BCR-ABL expression was not strongly affected by changes in cell cycle status. Taken together, these results provide the first evidence of multiple mechanisms of innate IM resistance in primitive and quiescent CML cells.

A novel tyrosine kinase switch is a mechanism of imatinib resistance in gastrointestinal stromal tumors

KIT or alpha-platelet-derived growth factor receptor (alpha-PDGFR) activating mutations are the pathogenic mechanisms that characterize gastrointestinal stromal tumors (GIST). Despite excellent responses to imatinib mesylate (IM), patients are relapsing. We developed an IM-resistant GIST cell line (GIST-R) from the IM-sensitive GIST882 cell line (GIST-S) by growing these cells in IM. Gene expression profiling (GEP) of GIST-S, GIST-R cells and two IM resistant GIST patients demonstrated that KIT is downregulated implying a major role in IM resistance. Instead, GIST-R cells have acquired IM resistance by overexpressing the oncogenic receptor tyrosine kinase - AXL - in a 'kinase switch'. Further, the two IM resistant GIST patients express AXL and not c-Kit, seen by immunohistochemistry (IHC). Real time reverse transcriptase-polymerase chain reaction and Western blotting of the GIST-S and GIST-R cells confirmed the switch from Kit to AXL. In GIST-R, AXL is tyrosine phosphorylated and its ligand growth-arrest-specific gene 6 is overexpressed implying autocrine activation. The kinase switch is associated with a morphological change from spindle to epithelioid. Molecular modeling of the kinase domain of mutant c-Kit (V654A) and AXL showed no binding to IM but efficient binding to MP470, a novel c-Kit/AXL kinase inhibitor. MP470 synergizes with docetaxel (taxotere) and is cytotoxic to GIST cells.

THE DUAL SRC/ABL KINASE INHIBITOR SKI-606 EFFECTIVELY INHIBITS BCR-ABL KINASE ACTIVITY IN CHRONIC MYELOGENOUS LEUKEMIA PRIMITIVE PROGENITOR CELLS.

Chronic myelogenous leukemia (CML) is a hematopoietic stem cell malignant disease that results from a translocation between chromosomes 9 and 22. The translocation generates the BCR/ABL gene, which has been shown to play a role in the pathogenesis of CML. Imatinib mesylate (IM), a Bcr-Abl kinase inhibitor, is highly effective in the treatment of CML. However CML stem cells appear to be relatively resistant to elimination by IM. Incomplete elimination of malignant progenitors may be related to incomplete Bcr-Abl kinase inhibition, persistent signaling through MAPK, or Bcr-Abl kinase mutations resulting in IM resistance. This phenomenon creates a need to develop new approaches that enhance elimination of the malignant progenitor cells. The dual Src/Abl kinase inhibitor SKI-606 has been reported to exert potent antiproliferative activity against CML cell lines in vitro and in xenograft models and is currently being investigated in phase 1/2 clinical trials. CD34+CD38− primitive progenitor and CD34+CD38+ committed progenitor cells from untreated CML patients were selected by flow cytometry and then cultured for 24 hours in growth factor-supplemented medium in a range of concentrations of SKI-606 (0-0.5 μM) and with 5 μM IM for comparison. The effect of SKI-606 on Bcr-Abl-kinase activity was assessed by Western blotting with anti-P-CrkL antibodies after overnight drug exposure of CML CD34+ cells. Importantly, 0.1 μM and 0.5 μM SKI-606 significantly suppressed phospho-CrkL levels, whereas a higher concentration of IM (5 μM) was needed to achieve a similar degree of suppression. Although treatment of CML CD34+ cells with IM was associated with increased p42/44 MAPK activity, a significant increase in MAPK activity was not observed when the same samples were treated with SKI-606. SKI-606 is therefore significantly more potent than IM in inhibiting Bcr-Abl TK in CML progenitors. Unlike IM, SKI-606 treatment was not associated with a significant compensatory increase in p42/44 MAPK signaling, which could potentially be beneficial in targeting malignant stem cells. Further investigation into the effects of SKI-606 on CML stem cells as a single agent or in combination with other compounds is warranted.

INNO-406, a novel BCR-ABL/Lyn dual tyrosine kinase inhibitor, suppresses the growth of Ph+ leukemia cells in the central nervous system, and cyclosporine A augments its in vivo activity

AbstractCentral nervous system (CNS) relapse accompanying the prolonged administration of imatinib mesylate has recently become apparent as an impediment to the therapy of Philadelphia chromosome–positive (Ph+) leukemia. CNS relapse may be explained by limited penetration of imatinib mesylate into the cerebrospinal fluid because of the presence of P-glycoprotein at the blood-brain barrier. To overcome imatinib mesylate–resistance mechanisms such as bcr-abl amplification, mutations within the ABL kinase domain, and activation of Lyn, we developed a dual BCR-ABL/Lyn inhibitor, INNO-406 (formerly NS-187), which is 25 to 55 times more potent than imatinib mesylate in vitro and at least 10 times more potent in vivo. The aim of this study was to investigate the efficacy of INNO-406 in treating CNS Ph+ leukemia. We found that INNO-406, like imatinib mesylate, is a substrate for P-glycoprotein. The concentrations of INNO-406 in the CNS were about 10% of those in the plasma. However, this residual concentration was enough to inhibit the growth of Ph+ leukemic cells which expressed not only wild-type but also mutated BCR-ABL in the murine CNS. Furthermore, cyclosporine A, a P-glycoprotein inhibitor, augmented the in vivo activity of INNO-406 against CNS Ph+ leukemia. These findings indicate that INNO-406 is a promising agent for the treatment of CNS Ph+ leukemia.

New Insights into the Pathophysiology of Chronic Myeloid Leukemia and Imatinib Resistance

Chronic myeloid leukemia (CML) was the first human malignant disease to be linked to a single, acquired genetic abnormality. Identification of the BCR-ABL kinase fusion protein and its central role in the pathogenesis of CML provided new opportunities to develop rational molecular targeted therapies. This review provides an update on the underlying pathophysiologies of disease progression and imatinib mesylate resistance, leading to the development of new targeted tyrosine kinase inhibitors for managing CML. Imatinib, a selective inhibitor of BCR-ABL, represents a major success in the era of target-directed cancer chemotherapy. However, patients with advanced CML have been less sensitive to therapy and responses have been short. In addition, treatment resistance is an emerging problem at all disease stages. Insight into factors involved in imatinib resistance and disease progression has highlighted a role for such BCR-ABL-dependent factors as amplification and overexpression of the BCR-ABL gene and the emergence of mutant isoforms of BCR-ABL. However, BCR-ABL-independent factors, including leukemogenic pathways involving kinases other than BCR-ABL, also play a part. In light of the limitations of imatinib against these factors, newer tyrosine kinase inhibitors, including dasatinib (a multitargeted kinase inhibitor of BCR-ABL and Src family kinases) and nilotinib (AMN107, a selective BCR-ABL inhibitor), may provide promising treatment options for patients with CML.

A Retrospective Survey of Imatinib Mesylate Treatment for Chronic Myeloid Leukemia in a Northern France Region.

Objective: To describe the use and results of imatinib mesylate (IM) treatment for chronic myeloid leukemia (CML) in the Nord-Pas de Calais Region in Northern France (~ 4 millions inhabitants).Methods: we identified all IM treated patients (pts) within the population of all confirmed diagnosis of CML occurred during the period 1985–2004. IM resistance (haematological and cytogenetic imatinib failure and suboptimal response) was evaluated according to the European LeukemiaNet consensus.Results: 302 pts (38.6% of the global cohort of diagnosis) were included in this retrospective study. If we consider new diagnosis occurred after Januar 1st 2001, 163 pts (90.6%) have been treated by IM. At the initiation of IM: 18 pts (6%) were in advanced phase (accelerated and blastic), 245 pts (81,1%) in chronic phase (CP) (with Ph+ metaphases > 35%), 35 (11,6%) had major cytogenetic response (MCyR) obtained with interferon (IFN) or bone marrow transplantation (BMT). Four pts (1,3%) had an unknown status. The percentage of alive pts at the last update (December 31th 2005) according to the disease stage at initiation of treatment is given in the table 1.In the sample of 245 CP pts, 139 (56.7%) had been previously treated by IFN, the median CML duration prior to IM was 4.7 months [0–174], the median IM treatment duration was 31.3 months [0.5–72.3] and the median follow-up after IM initiation was 36 month [5.2–72.3]. 214 pts (87.30%) were alive at the last update with 169 (79%) still on IM and 161 (75.2%) on IM as monotherapy. The median and mean IM daily doses of alive pts are 400 mg and 409.5 mg, respectively. Seventeen pts (6.9%) stopped IM for intolerance. The haematological and cytogenetic follow-up was informative for 206 CP pts (84.1%): MCyR 155 pts (75.2%) and CCyR 144 pts (69.9%). IM resistance occurred in 73 (35.4%) of these 206 evaluable pts, including 23 (11.1%) accelerations and blastic transformations. In the subgroup of IM resistant pts 47 (64.4%) have increased their IM posology. The mean of the maximum daily dose in the resistant subgroup was 607.5 mg (median 600 mg). BCR-ABL kinase domain mutations were found in 15 (20.5%) pts amoung IM resistant population. The estimated 5-years survival rate of the CP pts is 81.3%. Previous treatment by IFN doesn't influence significantly the survival, nor the duration of the disease before IM initiation, even if we found a trend for a better survival when this period was inferior to 6 months (5 years survival rate 90.4% vs 77.5%, p=0.16). BMT was performed for 31 pts (10.3%) including 19 pts (6.3%) after the initiation of IM therapy.Conclusion: these real life observations show the large use of IM in the last years and its great impact on the management and the survival of CML pts. IM resistance occurs in 1/3 of pts but the disease duration prior to IM is heterogeneous. An increase of IM dose has been tried in most IM resistant pts. The response to the different therapy adaptations is under study and will be presented. The vast majority of pts are still on IM therapy at the last update. [Display omitted]

Nilotinib Inhibits Bcr-Abl Kinase Activity in CML Progenitor Cells More Effectively Than Imatinib but Is Equipotent in Inducing Growth Inhibition.

The therapeutic success of imatinib mesylate (IM) in chronic myeloid leukemia (CML) is impaired by persistence of malignant stem cells. Mechanisms contributing to incomplete elimination of CML stem cells include resistance to proapoptotic effects of IM, persistent signaling through growth stimulating pathways and Abl kinase mutations resulting in IM resistance. We investigated whether nilotinib, a more potent Bcr-Abl tyrosine kinase (TK) inhibitor than IM, could more effectively target CML progenitors. CD34+ progenitors from CML patients and healthy donors were cultured with nilotinib (0–10μM) and IM (0–5μM) in growth factor (GF) containing medium. After 96h incubation, cells were harvested and assayed for colony-forming-(CFC) and long-term culture initiating cells (LTCIC). CD34+38- primitive progenitor cells (PPC) and CD34+38+ committed progenitor cells (CPC) labeled with CFSE were incubated under similar conditions and evaluated for proliferation and apoptosis assays by annexin-V staining and FACS analysis. The IC50 for CML LTCIC-suppression was 0.9μM for nilotinib and 0.8μM for IM. Normal LTCIC were not significantly suppressed at doses below 10μM nilotinib and 5μM IM. The IC50 for CML CFC suppression for nilotinib was 6.4μM and 1.0μM for IM. Normal CFC showed less growth inhibition and the IC50 was not reached at the dose range tested. CFSE assays indicated dose-dependent antiproliferative activity of both compounds with IC50-values of 3.0μM nilotinib and 1.8μM IM for CML PPC, and 8.2μM nilotinib and 4.0μM IM for CML CPC. In CML PPC, apoptosis significantly increased following nilotinib treatment (8±2.1% [control] to 22±2.7% [10μM], n=4, p=.015). IM treatment also increased apoptosis in CML PPC (to 35±3.8% [5μM], n=4, p=.0008). In CML CPC, apoptosis increased from 33±2.3% [control] to 50±1.7% (n=4, p=.002) with 10μM nilotinib, and to 53±2.1% (n=4, p=.001) with 5μM IM exposure. The effects on IM and nilotinib on Bcr-Abl-TK activity were investigated by Western blotting with anti-CrkL antibodies after overnight drug exposure of CML CD34+ cells in GF containing media. Importantly, 0.25μM nilotinib significantly reduced P-CrkL levels (from 85.7±4.1% [control] to 13.9±4.5%, n=4, p=.000002), whereas a higher concentration of IM (1–5μM) was needed to achieve similar inhibition. Nilotinib resulted in increased MAPK activity (to 13.3-fold [5μM]) in CML CD34+ cells. MAPK kinase activity was also increased following treatment with IM (to 22.7-fold [5μM]). P-AKT and P-STAT5 levels were not significantly changed in response to either drug. In contrast incubation with either drug in the absence of GF resulted in inhibition of MAPK, Akt and STAT5 activity in CML CD34+ cells. In conclusion, nilotinib is significantly more potent than IM in inhibiting Bcr-Abl TK in CML progenitors but does not induce greater suppression of progenitor growth. As with IM, inhibition of cell division represents the predominant mode of inhibition of progenitor growth. Nilotinib or IM treatment inhibits Bcr-Abl dependent MAPK, STAT5 and Akt activity, but does not inhibit these signaling mechanisms in CML progenitors in the presence of GF. Our results suggest that combined inhibition of Bcr-Abl TK and additional signaling mechanisms may be required to achieve elimination of CML progenitors by targeted therapies.

Imatinib Resistance Restoration with Antioxidants and Immunomodulation in Patients with Chronic Myelogenous Leukemia (CML).

Imatinib mesylate (IM) is potent BCR/abl tyrosine kinase inhibitor in patients with chronic myelogenous leukemia (CML). It has remarkable frontline clinical effects in this disease, however, the leukemic cells become resistant to IM in both chronic and blast phases. BCR/abl kinase can induce reactive oxygen species (ROS) and promote self-mutation which subsequently render IM to resistance and failure to eliminate all leukemia cells. This mechanism of resistance in IM (mutation) is caused by oxidant damage to DNA with kinase domain mutations, reduced IM binding and kinase inhibition. Antioxidants (ascorbic acid, etc.) may help overcome IM resistance and restore sensitivity to IM via suppression of transcription factor Nrf2 that regulates the gene expression gammaglutamylcysteine ((g-GCS), the rate-limiting enzyme in glutathione (GSH) biosynthesis and detoxification. P-glycoprotein (P-gp) drug efflux can also exist and complete molecular response relapse occurs. Leukemic cells that are P-gp positive, and P-gp dependent decline of intracellularIM levels are associated with retained phosphorylation pattern of BCR/abl and loss of IM effect on apoptosis and cellular proliferation. Modulation of P-gp with HMG-CoA reductase inhibitor simvastatin may help restore IM cytotoxicity. We present a case of an 80+ year old female with CML-chronic phase-II (CML-CP-II) with concommitant cormorbidities of CAD, unstable angina, hypertension, and dyslipidemia treated with aspirin, simvastatin and started on IM 400mg daily. After two months of therapy she developed grade 3 neutropenia, lower extremity edemia, nausea/vomiting and fluid retention requiring IM interruption and supportive care with growth factors. IM dose reduction to 300mg daily and simvastatin 10 mg every other day. Soluble interleukin-2 receptor (sIL-2R) levels were elevated and trending down once proinflammatory cytokines were modulated. Real-time BCR-ABL/abl-PCR ratio increased insignificantly from 0.001% to 0.003%. Betacarotene level significantly decreased to 4, ascorbic level within normal limits, VEGF remained < 31 pg/ml (normal 31–86 pg/ml), fibrinogen level 309.90 mg/dl (normal 162–431), ESR 15 mm/hr, C-reactive protein 5.55 mg/dl and sIL-2R increased to > 3,000 U/mL (normal 200– 1100 U/mL). Betacarotene and ascorbic antioxidants dosage were increased and immunomodulation of preinflammatory cytokines ROS, sIL-2R and betacarotene normalized, 512 and 44 respectively. Serial measurement of BCR-ABL/abl ratio did not exceed 0.02% on three occasions or 0.05% on two occasions, therefore no molecular relapse and persistent low levels of BCR-ABL/abl ratio with no hematologic or cytogenetic relapse. We felt that an acute coronary syndrome with perturbation of CML with some IM resistance was developed.CONCLUSION: IM, a tyrosine kinase inhibitor may develop resistance when leukemic cells are positive with P-gp and oxidative stress increase ROS along with decreases in IM intracellular levels. Modulation by HMG -CoA reductase inhibitor (simvastatin) via a mechanism of inhibition of P-gp transport and antioxidants reduction of BCR/abl mutagenesis may allow IM to efficently restore normal hematopoiesis in CML patients.

The Dual Src/Abl Kinase Inhibitor SKI-606 Effectively Inhibits Bcr-Abl Kinase Activity and Reduces Proliferation of CML Primitive Progenitor Cells.

Imatinib mesylate (IM) is highly effective in the treatment of CML. However resistance to IM can develop in a subset of patients. In addition CML stem cells appear to be relatively resistant to elimination by IM. Incomplete elimination of malignant progenitors may be related to incomplete Bcr-Abl kinase inhibition, persistent signaling through MAPK or other growth stimulatory pathways, or Bcr-Abl kinase mutations resulting in IM resistance. The dual Src/Abl kinase inhibitor SKI-606 has been reported to exert potent antiproliferative activity against CML cell lines in vitro and in xenograft models, and is currently being investigated in phase1/2 clinical trials. Here, we investigated whether SKI-606 could effectively target CML primary progenitors. Flow cytometry selected CD34+CD38− primitive progenitor (PPC) and CD34+CD38+ committed progenitor cells (CPC) from untreated CML patients were cultured for 96h in growth factor (GF) supplemented medium in a range of concentrations of SKI-606 (0–0.5μM), and with 5μM IM for comparison. Cells were labelled with CFSE-prior to culture and with Annexin-V at culmination of culture to allow flow cytometry assessment of the effects of drug exposure on cell proliferation and apoptosis. In addition CD34+ cells were similarly incubated with SKI-606 and subsequently plated in methylcellulose progenitor culture to assess effects on colony forming cell (CFC) growth. CFSE assays indicated significant dose dependent antiproliferative activity of SKI-606 with IC50-values of 0.2μM for CML PPC. SKI-606 resulted in moderate induction of apoptosis of CML PPC (from 22±7.2% [control] to 49±16.4% [0.5μM], n=3, ns). CFC-assays consistently revealed significant dose dependent growth inhibitory effects of SKI-606 with IC50-values of 0.1μM SKI-606. The effect of SKI-606 on Bcr-Abl-kinase activity was assessed by Western blotting with anti-P-CrkL antibodies after overnight drug exposure of CML CD34+ cells. Importantly, 0.1μM and 0.5μM SKI-606 significantly suppressed phospho-CrkL levels (from 94.7±3.5% [control] to 14.9±4.8%, n=4, p=.0001, and to 6.8±2.5%, n=4, p=.00003, respectively), whereas a higher concentration of IM (5μM) was needed to achieve a similar degree (13.1±5.0%, n=4, p=.0000007). Whereas treatment of CML CD34+ cells with IM was associated with increased p42/44 MAPK activity (n=3, p=.038), a significant increase in MAPK activity was not observed when the same samples were treated with SKI-606 (n=3, ns). In conclusion, SKI-606 significantly inhibits CML progenitor proliferation and moderately induces apoptosis at lower concentrations than previously observed with IM, although the maximum growth suppression effects observed were not greater than those observed with high concentrations of IM. SKI-606 is significantly more potent than IM in inhibiting Bcr-Abl TK in CML progenitors. Unlike IM, SKI-606 treatment was not associated with significant compensatory increase in p42/44 MAPK signaling, which could potentially be beneficial in targeting malignant stem cells. Further studies investigating the effects of SKI-606 on CML stem cells as a single agent or in combination with other compounds are warranted.

Overexpression of the ABC-Transporters BCRP and MDR1 in CML Patients with Resistance to Imatinib and in Untreated CML Patients.

Imatinib Mesylate (Gleevec™ or Glivec™ from NOVARTIS) is widely used for the treatment of CML inhibiting the BCR-ABL tyrosine kinase activity. Although, 90% of the patients respond to this treatment, resistance to this drug has been seen, mainly due mutations in the tyrosine kinase domain of BCR-ABL. Recently, has been reported that imatinib is a substrate for the ABC-transporters BCRP (breast cancer resistance protein) and MDR1 (P-Glycoprotein). Based in that report, we examined the expression of those two ABC-transporter, by quantitative PCR (Q-PCR) technology, in 29 CML patients who were resistant to IM (9 CP, 17 AP, and 3 BC), in 34 untreated CML patients (23 CP, 10 AP, and 1 BC) and 6 normal blood donors. cDNA was synthesized for RQ-PCR, which was performed on peripheral blood samples drawn after loss of CHR or MCyR for those with resistance to imatinib and prior to start of treatment with imatinib and those untreated patients. All samples were analyzed using SYBER Green technology and reported as the 2−Δ Δ Ct where it assumes that the amplification efficiency of the target gene (BCRP or MDR1) and the internal control gene (ABL) are the same. Resistant patients had a significantly higher levels of BCRP expression than untreated patients, and normal blood donors with a median ratio of 2.1 (range, 0.04–34.2) vs. 0.64 (range, 0–18.6) vs. 0.6 (range, 0.07–1.41) (Wilcoxon test, P<0.0002). For the MDR1 gene expression, patients with resistance to imatinib had also a significantly higher level, median ratio of 1.65 (range, 0.09–10.8) vs. 0.68 (range, 0.01–2.63) in untreated patients and normal blood donors median ratio 0.76 (range, 0.02–1.92) (P < 0.0017). Nineteen of 29 resistant patients (65.5%) overexpressed BCRP and 41.3% (12/29) overexpressed MDR1, meantime only 26.5% (9/34) and 11.8% (4/34) of the untreated CML patients overexpressed BCRP and MDR1 respectively (X2 test P=0.004 and P=0,022). Overexpression of BCRP was more frequent in AP of the resistant group (70%) than the untreated patients (30%) (Fisher exact test P=0.05). When comparing the expression of MDR1 between the resitant group in AP (35,3%) and the untreated one in the same phase (40%) no statistic difference was seen (P=1). Patients in CP and BC do not show any difference in the expression of BCRP and MDR1 regardless the presence or not of resistance to imatinib. These data confirms that high expression of BCRP or MDR1 may contribute to imatinib resistance phenotype leading to a reduction in intracellular imatinib concentrations.Further follow-up is necessary for those untreated CML patients that overexpress, at diagnosis, those ABC-transporters BCRP and MDR1. These may reveal correlations between the expression and clinical prognosis and may allow the development of new starategies to overcome imatinib mesylate resistance.

Role of BCR/ABL Gene Expression Levels in Determining the Phenotype and Imatinib Sensitivity of Transformed Human Hematopoietic Cells.

Increased levels of BCR/ABL expression in chronic myelogenous leukemia (CML) cells is associated with disease progression from chronic phase (CP) to blast crisis (BC), and resistance to imatinib mesylate (IM) treatment. However, it is not clear if these associations directly result from elevated BCR/ABL expression levels or reflect coincidental occurrence of additional abnormalities. Here we used an experimental model of human CML based on ectopic expression of the BCR/ABL gene in human CD34+ cells to directly investigate the role of increased BCR/ABL expression levels in determining the phenotype of transformed hematopoietic cells and their IM response. CD34+ cells were transduced with retroviral vectors expressing BCR/ABL and GFP. Cells with low and high levels of GFP and BCR/ABL expression (BAlo and BAhi) were selected by flow cytometry. We confirmed increased expression of BCR/ABL in GFP high cells by quantitative RT-PCR and Western blot analysis. The average BCR/ABL expression levels in BAlo cells (0.51 ± 0.19, n=3) were consistent with those observed in CD34+ cells from CML CP (0.36 ± 0.11, n=4) and accelerated phase (AP) patients (0.62 ± 0.08, n=3), and in BAhi (0.95 ± 0.09, n=3) cells with those observed in CML BC patients (0.98 ± 0.09, n=3). BAhi cells demonstrated significantly higher expansion after growth factor (GF) culture compared with BAlo cells (expansion of control cells: 14.3 ± 3.7, BAlo: 33.7 ± 9.3 and BAhi: 65.4 ± 16.4, n=4). Increased expansion was related to both increased cell division and reduced apoptosis. The proliferation rate of BAhi cells, measured by labeling cells with the fluorescent dye SNARF-1, was significantly increased compared with BAlo cells (proliferation index for controls: 5.3 ± 1.0, BAlo: 24.3 ± 8.1, and BAhi: 41.8 ± 11.9, n=4). BAhi cells demonstrated increased resistance to apoptosis upon GF deprivation, (28 ± 6.7%, n=5) compared with BAlo (45 ± 8%, n=5) or control cells (56 ± 9.3%, n=5). Interestingly, increased BCR/ABL expression levels in human hematopoietic cells was associated with enhanced IM sensitivity rather than with IM resistance (IC50 for BAhi = 0.025 μM; BAlo = 0.2 μM), related to both increased inhibition of proliferation (% inhibition of 55 ± 4.3% for BAhi, vs. 35.5 ± 4.4% for BAlo cells with 0.1 μM IM, n=4) as well as increased induction of apoptosis (% increase in apoptosis with 0.1μM IM for BAhi 26.4 ± 4% vs. BAlo 8.4 ± 3 %, n=5). We did not observe a significant difference in phosphorylation of AKT and MAPK between BAhi and BAlo cells, but did observe significantly increased STAT5 phosphorylation in BAhi CD34+ cells compared to BAlo cells (pSTAT5:STAT5 for BAlo = 0.41 ± 0.21, BAhi = 1.01 ± 0.1). Furthermore, electromobility shift assays (EMSA) confirmed increased STAT5 promoter activity in BAhi cells compare to BAlo cells. In conclusion, increased levels of BCR/ABL expression in human CD34+ cells results in increased expansion in hematopoietic cell numbers related to enhanced proliferation and increased resistance to apoptosis. However expression of high levels of BCR/ABL was associated with enhanced rather than reduced sensitivity to IM. These observations suggest that BCR/ABL overexpression may contribute to disease progression in CML but may not play a direct role in IM resistance in cells overexpressing BCR/ABL. Our results suggest a role of increased STAT5 activity in abnormal growth of BCR/ABL overexpressing cells.