BCR-ABL Tyrosine Kinase Activity Research Articles

Targeting BCR-ABL and Its Downstream Signaling Cascade as Therapy for Chronic Myelogenous Leukemia.

BCR-ABL is an unregulated tyrosine kinase expressed as a consequence of chromosomal translocation in chronic myelogenous leukemia (CML). The tyrosine kinase activity of BCR-ABL activates signaling cascades that induce cytokine independence and transformation of myeloid progenitor cells. Targeted inhibition of this kinase with specific inhibitors (imatinib or BMS-354825) is a very effective therapy for some CML patients but resistance to these agents (through point mutations and other mechanisms) leads to advanced disease with very few therapeutic options. An alternate therapeutic strategy is to reduce BCR-ABL expression or its critical downstream signaling elements important for transformation. We examined BCR-ABL signaling elements and gene expression changes that occur in CML cells following kinase inhibition by imatinib in newly established imatinib sensitive and resistant cells to identify critical signaling elements involved in CML cell death. Imatinib rapidly and progressively suppressed c-myc expression in imatinib sensitive but not resistant cells prior to the onset of apoptosis. These results suggested that c-myc expression was regulated by BCR-ABL signaling and may play a role in CML tumorigenicity. To confirm a role for c-myc in CML cell growth and/or survival, c-myc expression was specifically down-regulated by siRNA using a novel electroporation instrument (AMAXA) that permits high level gene transfer with limited toxicity in CML cell lines. Jak2 siRNA was used as a control. c-myc, but not Jak2 siRNA, suppressed c-myc expression and cell growth and survival in both imatinib sensitive and resistant CML cells, suggesting that targeted suppression of c-myc may have therapeutic activity against both kinase inhibitor sensitive and resistant CML cells. Since the tyrphostin AG490 was previously shown to inhibit c-myc expression in CML cells through its inhibitory effects on Jak2, we screened a series of > 200 AG490 derivatives for their ability to rapidly reduce c-myc expression in hematological malignancies. After several rounds of testing we synthesized an agent (WP-1066) capable of rapid c-myc downregulation (beginning 1–5 min after treatment with 1–2 microM) but poor Jak2 kinase inhibitory activity (IC50 > 100 microM). These results suggested a more direct effect of WP-1066 on c-myc protein expression than AG490 and mechanistic studies suggest that WP-1066 reduces c-myc protein stability but does not affect c-myc gene expression. In BCR-ABL expressing cells WP-1066 rapidly reduced c-myc protein levels in CML cells and inhibited the growth and survival of cell lines or patient specimens expressing wild-type or mutant forms of BCR-ABL that effect tyrosine kinase inhibitory activity (T315I in BV-173R cells). Equal concentrations of imatinib or WP-1066 reduced BCR-ABL activation and downstream signaling (Stat5 phosphorylation) in CML cells. However, WP-1066 differed from imatinib in its ability to downregulate BCR-ABL protein expression without affects on c-abl or Stat5 expression. Similar results were obtained in clinical specimens taken from patients with BCR-ABL point mutations that mediate imatinib (or BMS-354825) resistance. Nude mouse studies demonstrated that WP-1066 reduced the growth of K562 tumors to an extent similar to that of imatinib. Together these results suggest that WP-1066 downregulates BCR-ABL and c-myc expression, induces apoptosis in CML cells expressing wild-type or mutant BCR-ABL and may have therapeutic activity in imatinib (or BMS-354825) resistant CML tumors.

Bim-EL, Is Sensitive to Proteasome Degradation and Contributes to Inducing Apoptosis after In Vitro Imatinib Mesylate Treatment of BCR-ABL Positive Cells.

Chronic myeloid leukemia (CML) is a malignant disease of pluripotent hematopoietic stem cells and is characterized by excessive accumulation of the myeloid lineage. The causative event in the initiation of CML is the translocation of the abl gene in continuity with the bcr gene resulting in a fusion gene encoding for a Bcr-Abl tyrosine kinase. The expression of Bcr-Abl protects hematopoietic cells against apoptosis. We investigated the survival pathways triggered by Bcr-Abl. Apoptosis was measured in two cell lines expressing the Bcr-Abl protein either constitutively (K562) or ectopically (BaF/Bcr-Abl). Imatinib mesylate, an inhibitor of the tyrosine kinase activity of Bcr-Abl, induced apoptosis and caspase activation in K562 and BaF/Bcr-Abl and this effect was almost completely inhibited by cycloheximide (CHX) indicating that imatinib needs de novo protein synthesis to induce apoptosis in Bcr-Abl cell lines. MG132, a proteasome inhibitor, was also found to induce apoptosis in both cell lines, an effect also inhibited by CHX. By measuring mitochondrial membrane potential we found that CHX affected the apoptotic process induced by both inhibitors upstream of the mitochondrial step. Co-treatment with imatinib and MG132 failed to show any additive effect and imatinib did not inhibit proteasome activity by itself. These results are in agreement with Bcr-Abl tyrosine kinase exerting its anti-apoptotic effect by driving the degradation of a pro-apoptotic protein with a high turnover which, after phosphorylation, became a substrate for the proteasome. When protein kinase C was activated by PMA, Bim-EL, a pro-apoptotic, BH3-only member of the Bcl-2 family, was converted in its phosphorylated form and its cell content was decreased by 60%. The phosphorylated form of Bim-EL was preserved when proteasome was inhibited by MG132 inducing a two times increase in Bim-EL protein content. Similarly, the content in Bim-EL was increased (three times in K562 and five times in BaF/Bcr-Abl) after 18h imatinib treatment. In this case, Bim-EL accumulated in an un-phosphorylated form and this accumulation was inhibited by CHX. Thus, Bcr-Abl kinase could indirectly drive the phosphorylation of Bim-EL, maintaining its degradation by the proteasome and, consequently, a low apoptotic potential for the cell. Conversely, Bim-EL accumulation could play a crucial role in the apoptotic response of Bcr-Abl expressing cells treated by either imatinib or proteasome inhibitors.

AMD107: Efficacy as a Selective Inhibitor of the Tyrosine Kinase Activity of BCR-ABL in Murine Leukemia Models.

Patient relapse due to the emergence of imatinib-resistance in advanced chronic myelogenous and Ph+ acute lymphocytic leukemias has prompted the search for improved Bcr-Abl kinase inhibitors. Up to 40% imatinib-resistant patients express mutant forms of the Bcr-Abl kinase that reduce binding of imatinib without significantly decreasing kinase activity. However, some mutants are only partially resistant, since higher concentrations of imatinib may still block kinase activity and have a beneficial clinical effect. More potent Bcr-Abl inhibitors or inhibitors with different contact points with the kinase domain are predicted to be useful for the treatment of imatinib-resistant disease. Rational drug design based upon the crystal structure of an Abl-imatinib complex, together with medicinal chemistry paradigms, resulted in the discovery of AMN107. AMN107 is a low molecular weight compound that selectively inhibits the c-Abl/Bcr-Abl, imatinib-resistant mutants of Bcr-Abl, PDGFR, and c-Kit tyrosine kinases, with IC50 values for the inibition of autophosphorylation (cell capture ELISA) of 23 nM (32D cells transfected with p210 Bcr-Abl), 83 nM (A31 cells) and 192 nM (GIST cells), respectively. Following oral administration to mice (20 mg/kg in NMP-PEG300 10:90 v/v), AMN107 was well absorbed, with a mean plasma level after 2 h in the range of 6.0-12.1 M, which corresponds to >100-fold that required to inhibit Bcr-Abl autophosphorylation in 32D.p210 cells. Following injection of 32D.p210-luciferase cells into Balb/c mice, serial imaging of the leukemic clone in live mice indicated that AMN107 could substantially reduce the accumulation of leukemic cells in the marrow, nodes, liver, and spleen, compared to a vehicle control. Further, administration of AMN107 (75 mg/kg/day p.o.) to BALB/c mice over an 18 day period, commencing 3 days after the injection of 32 D.p210 cells, resulted in the protection of 15/20 animals over 100 days observation, whereas 19/20 vehicle treated animals developed a lethal leukemic disease(all deaths occurred within 36 days of cell injection). The spleen sizes of the surviving animals in the treated group were in the normal range (0.142±0.043 g; n = 17), whereas those of the vehicle treated animals were 0.50±0.045 (n = 20). These results were confirmed in a bone marrow transplant assay, where AMN107 was found to prolong survival of mice transplanted with marrow cells infected with a p210Bcr/Abl retrovirus. AMN107 also prolonged survival of mice transplanted with a Bcr/Abl mutant (E255V) associated with imatinib resistance in patients, while imatinib treatment was unsuccessful. These in vivo studies, along with in vitro studies reported separately, indicate that AMN107 is a highly active inhibitor of Bcr/Abl that may have clinical utility in patients with Bcr/Abl+ leukemias.

AMN107, Novel Aminopyrimidine Inhibitor of Bcr-Abl, Is Significantly More Potent Than Imatinib Mesylate Against Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Cells.

Imatinib mesylate is effective against Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL) but, when used as a single agent, responses are transient and most patients relapse within 4–6 months. AMN107 is a novel oral aminopyrimidine ATP-competitive inhibitor of the protein tyrosine kinase activity of Bcr-Abl. Following oral administration to animals, AMN107 is well absorbed, has a good pharmacokinetic profile, and is well tolerated. The activity of AMN107, relative to imatinib, in both Ph-positive (Z-119 and Z-181) and Ph-negative (Z-138) ALL cell lines was studied. Z-119 and Z-181 cells were derived from Ph-positive ALL patients and retained typical B-cell characteristics and phenotypes of the original leukemia, including cytogenetic abnormality t(9;22) and p190 Bcr/Abl kinase. Z-138, a Ph-negative cell line, was derived from a patient with chronic lymphocytic leukemia and supervening ALL. Treatment with AMN107 or imatinib for 3 days (MTS assay) inhibited proliferation of Z-119 cells with the IC50 values of 19.3 nM and 620.0 nM, respectively, revealing AMN107 to be 32 fold more potent than imatinib. Treatment of Z-181 cell line lasted for 4 days (MTS assay) because of lower growth rate of these cells: IC50 for AMN107 and imatinib were 1.6 nM and 63.9 nM, respectively, showing AMN107 to be 40 fold more potent than imatinib. Neither drug showed activity against Ph-negative Z-138 cells. We also compared the activity of AMN107 in Ph-positive ALL cell lines expressing p190 Bcr/Abl protein to that in Ph-positive chronic myeloid leukemia cell lines KBM5 and KBM7 expressing p210 Bcr/Abl protein. The activity was similar with IC50 in KBM5 cells of 11.3 nM and in KBM7 cells of 4.3 nM. In experiments focused on cell cycle analysis we found that at equipotent doses (as determined by MTS assay) both drugs induced cell accumulation in G0/G1 phase in Z-119 but not in Z-181. We demonstrated that increasing equipotent concentrations of AMN107 and imatinib induced activation of caspase-3 that resulted in apoptosis, as assessed by propidium iodide staining, in Z-119 cells, while Z-181 cells showed lack of apoptotic response. Following treatment with a broad range of AMN107 and imatinib doses for 3 hrs, Bcr/Abl expression and phosphorylation were determined in Z-119 cells by immunoprecipitation and Western blotting: Bcr/Abl phosphorylation was inhibited completely with AMN107 at 125.0 nM, and with imatinib at 2500 nM, confirming again the higher potency of AMN107. Finally, similar differential effect of AMN107 and imatinib on Bcr/Abl protein expression and phosphorylation was observed in leukemic cells obtained from blood of Ph-positive ALL patients. We conclude that AMN107 has significant activity against Ph-positive ALL cells and warrants investigation in patients with Ph-positive ALL.

Combined Effects of As4S4 and Imatinib on Chronic Myeloid Leukemia Cells and BCR-ABL Oncoprotein.

Imatinib is a tailored drug for chronic myeloid leukemia (CML), which has very good effects on patients at chronic phase (CP), but not on those at accelerated phase or blast phase. In addition, even among patients at CP, Imatinib seems unable to eradicate the malignant progenitors and a significant portion of patients develops drug resistance after long time use. Arsenic compounds were known as ancient remedies for CML with certain efficacy. The aim of this study was to investigate the potential benefit of combination therapy with Imatinib and arsenic sulfide (As4S4) on BCR-ABL+ K562 cells and fresh CD34+ hematopoietic progenitor cells isolated from CML patients and non-leukemic donors. Analysis of cell proliferation and clonogenic ability showed that As4S4 and Imatinib exerted synergistic effects on both K562 cells and fresh CML cells. The effective concentrations on fresh CML cells were pharmacokinetically available in vivo but had much less inhibitory effect on CD34+ cells from the non-leukemia donors. The synergistic effect of Imatinib/As4S4 combination in terms of anti-proliferation might be connected with their distinct but complementary roles in interfering with the cell cycle progression. Our data showed that Imatinib induced G1 arrest of K562 cells, while As4S4 induced G2/M arrest. In addition, Imatinib induces significant down-regulation of phosphorylated Rb and CDK1, which is in agreement with the G1/S but not G2/M arrest under this drug. However, As4S4 shows no obvious effect on these proteins in spite of a visible effect on G2/M block. Using a number of parameters such as morphology, Annexin V/PI, mitochondrial transmembrane potential, caspase3 activity and Fas/Fas-L, the synergistic effects were revealed on induction of cell apoptosis, largely through mitochondrial pathway. What's more, the two drugs also exhibited synergistic effect in targeting BCR-ABL protein. While As4S4 triggered its degradation and Imatinib inhibited its tyrosine kinase activity, combined use of the two led to lower protein/enzymatic activity levels of BCR-ABL. In conclusion, our study suggests As4S4 and Imatinib have synergistic effects in inhibiting proliferation, inducing apoptosis of cells and reduction the tyrosine kinase activity of BCR-ABL. Our in vitro data thus strongly suggest a potential clinical application of Imatinib/As4S4 combination on CML.

AMN107: In Vitro Profile of a New Inhibitor of the Tyrosine Kinase Activity of Bcr-Abl.

Imatinib mesylate (Gleevec®, Novartis Pharma AG), an inhibitor of the c-Abl/Bcr-Abl, c-Kit, and PDGFR kinases, is an effective therapy for CML, GIST and HES. However, advanced stage CML and Ph+ ALL patients frequently relapse due to the development of resistance. In up to 50% of cases, imatinib resistance is the result of the clonal expansion of cells expressing Bcr-Abl kinase domain mutants and more than 30 such mutations have been detected. Different mutations display varying degrees of resistance, and although some are sensitive to increased concentrations of imatinib, new Abl kinase inhibitors with higher potency against wild-type and imatinib-resistant mutants of Bcr-Abl could have substantial clinical utility. Rational drug design based on the structural information obtained from the crystal structure of the Abl-imatinib complex and medicinal chemistry optimization of drug-like properties, resulted in the discovery of AMN107. The potencies of AMN107 and imatinib were compared with cellular assays using CML lines (K562 and KU812F) and Ba/F3 cells expressing either ‘wt'- or imatinib-resistant mutants of Bcr-Abl. AMN107 effectively reduced the cellular Bcr-Abl autophosphorylation in K562, KU812F and Ba/F3 Bcr-Abl cells with IC50s of 42, 60 and 23 nM, respectively (Gleevec: 470, 399 and 231 nM) The proliferation of these cells was inhibited with IC50s of 11, 8 and 23 nM (Gleevec: 272, 80 and 634 nM). Autophosphorylation of the Bcr-Abl mutants M351T, F317L and E255V was reduced by AMN107 with IC50s of 33, 43 and 245 nM (Gleevec: 595, 818 and 6499 nM), resulting in inhibition of cell proliferation with IC50s of 30, 77 and 684 nM (Gleevec: 1285, 1583 and 6294 nM). No effect could be observed below 8 μM against the T315I and G250E bcr-abl mutants. Similar to imatinib, AMN107 inhibited the cellular tyrosine kinase activity of PDGFR-β (A31 cells) and c-Kit (GIST882 cells), with IC50s of 85 and 192 nM (Gleevec: 74 and 96 nM). Control (Ba/F3) cell proliferation was unaffected by 10 μM AMN107, and added IL-3 abrogated the antiproliferative effects on Bcr-Abl expressing Ba/F3 cells. Furthermore, AMN107 (< 10 μM), did not effect unrelated kinases, e.g. c-erbB2 (BT474), Ins-R (A14) and IGF-1R (NWT-21), in cellular assays, consistent with its specificity and lack of general cytotoxicity. Based on these in vitro assays and preclinical in vivo and pharmacokinetic data to be presented separately, AMN107 represents a promising new tyrosine kinase inhibitor for testing in human clinical trials.

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

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.

Differential Regulation of Pro- and Anti-Apoptotic Genes by Bcr-Abl in an In Vitro Experimental Model of Chronic Myelogenous Leukemia.

Leukemic cells from patients with Philadelphia chromosome-positive chronic myelogenous leukemia (CML) are very resistant to apoptosis induced by DNA-damaging agents and other chemotherapeutic drugs, due to the presence of Bcr-Abl, a chimeric cytoplasmic tyrosine-kinase that confers both malignancy and resistance to apoptosis. Efficient treatment of CML can be achieved with a normal bone marrow transplant, which induces a graft-versus-leukemia response, and more recently by the use of the specific inhibitor imatinib mesylate (glivec. Novartis). Glivec blocks Bcr-Abl kinase activity and, as a consequence, the malignant cell dies by apoptosis. However most glivec-treated patients, mainly in the acute and blast phases, develop resistant forms of the disease. Since resistance to apoptosis in Bcr-Abl+ cells is probably related to the inhibition of mitochondrial release of cytochrome c, an obligatory step in most apoptotic pathways, we sought to investigate expression of Bcl-2 family genes in Bcr-Abl+, glivec-treated cells. By semi-quantitative RT-PCR we analyzed the gene expression of several pro- and anti-apoptotic molecules in the transduced cell line HL-60.Bcr-Abl and the wild-type HL-60, after a 1, 4 and 8h treatment with 10μM glivec. Bcr-Abl′s kinase activity is promptly inhibited by glivec (within 5 to 15min) and HL-60.Bcr-Abl cells begin to show mitochondrial depolarization 24h after treatment with the drug, dying 48h later, whereas no effects are observed in HL-60. Soon after glivec addition some genes are transcriptionally regulated in HL-60.Bcr-Abl cells. The major differences were observed for bcl-xL (2-fold reduction), c-flip (2-fold increase), bcl-w (30% increase) and mcl-1 (20% reduction). Some pro-apoptotic molecules such as noxa also displayed differential regulation in HL-60.Bcr-Abl cells. No differences were observed in HL-60 cells. In conclusion we describe a complex transcriptional regulation mechanism dependent on Bcr-Abl tyrosine-kinase activity, which has not been previously described by the use of microarrays, and could contribute to the understanding of the mechanisms involved in protection of apoptosis and drug resistance of Bcr-Abl+ cells.

Synergistic Cytotoxic Effects of a Combination of a Novel Tyrosine Kinase Inhibitor AMN107 and Histone Deacetylase Inhibitor LBH589 Against Bcr-Abl Expressing Human Leukemia Cells.

AMN107 (Novartis Pharmaceuticals, Basel, Switzerland) is a potent aminopyrimidine tyrosine kinase (TK) inhibitor, which is active at nanomolar concentrations against c-Kit, PDGFR as well as the wild type Bcr-Abl and common variety of mutant Bcr-Abl (e.g., M351T, F317L and E255V) TK. In a previous report, we demonstrated that treatment with the hydroxamic acid analogue histone deacetylase inhibitor LBH589 (Novartis) (20 to 100 nM) induces hsp90 acetylation and promotes polyubiquitylation and proteasomal degradation of Bcr-Abl, which is associated with apoptosis of the cultured Bcr-Abl expressing human chronic myeloid leukemia-blast crisis (CML-BC) K562 and LAMA-84 cells, as well as of primary CML-BC cells. In the present studies, we determined the cell cycle and apoptotic effects of AMN107 and/or LBH589 in K562, LAMA-84 and primary CML-BC cells. Treatment with AMN107 (20 to 100 nM) induced cell cycle G1 phase accumulation and apoptosis, exerting 10 to 20 fold more potent effect than imatinib mesylate (IM) in K562 and LAMA-84 cells. This was associated with marked induction of p27, inhibition of Bcr-Abl TK activity, as well as attenuation of the levels of p-STAT5, p-AKT, Bcl-xL, and c-Myc levels in a dose dependent manner. Co-treatment with LBH589 (50 nM) and AMN107 (100 nM) induced more G1 phase accumulation than either agent alone. The combination of AMN107 and LBH589 also exerted synergistic apoptotic effects in K562 cells, as determined by the median effect isobologram analysis. This was associated with more attenuation of the levels of pro-growth and pro-survival proteins, e.g., p-STAT5, p-ERK1/2, c-Myc and Bcl-xL, as well as greater induction of the levels of pro-death p27 and Bim proteins. Treatment of human AML HL-60 cells containing ectopic expression of the IM-refractory, mutant Bcr-AblT315I with LBH589 (50 nM for 24 hours) attenuated the mutant Bcr-Abl levels and induced apoptosis of HL-60/Bcr-AblT315I cells. Treatment with AMN107 (up to 2.0 μM) alone was ineffective in inducing loss of viability of HL-60/Bcr-AblT315I cells, and co-treatment with LBH589 (50 nM) and AMN107 did not induce more loss of cell viability of HL-60/Bcr-AblT315I cells than treatment with LBH589 alone. AMN107 levels ≥ 200 nM significantly inhibited survival of HL-60/Bcr-AblE255K and HL-60/Bcr-AblM351T cells. Consistent with this, the combined treatment with LBH589 and AMN107 also induced more loss of viability than treatment with either agent alone in 4 samples of IM-refractory primary CML-BC cells. These studies demonstrate that both AMN107 and LBH589 are active against IM-resistant CML-BC and the combination of the two exerts superior activity against IM-sensitive and IM-resistant, wild type and mutant Bcr-Abl expressing cultured and primary CML cells.

Activity and Induction of Apoptosis of the Specific Tyrosine Kinase Inhibitor AMN 107 in bcr-abl + Cell Lines and in Imatinib Resistant Primary Cells from CML Patients.

The selective tyrosine kinase inhibitor imatinib eradicates bcr-abl+ cells in chronic myeloid leukemia patients (pts). Although a previous clinical trial showed superiority of an imatinib therapy over an interferon-α containing regimen, a significant number of pts eventually relapse with leukemia because of either point mutations within the imatinib-binding site, amplification of the Philadelphia chromosome or other mechanisms, e.g. clonal evolution. AMN107 (Novartis Pharma AG) is a new anilino-pyrimidine derivative (MW: 529.5) structurally related to imatinib.AMN107 was tested in three human bcr-abl positive lines (K562, KCL-22, Lama-84) and in primary cells derived from two bcr-abl + CML pts who were resistant to imatinib, as well as in one newly diagnosed chronic phase patient. In all pts sequencing of the bcr-abl kinase domain excluded any point mutations, but cytogenetic analysis of the bone marrow revealed clonal evolution in the resistant pts including t(1;5) and t(3;21) translocations, trisomy of chromosome 8 and monosomy of chromosome 7.Determination of the proliferative activity by XTT-assay in cell lines demonstrated a decrease of the IC50 in imatinib versus AMN107 treated samples from 0.08μM to 0.0075μM in Lama 84, from 0.25μM to 0.08μM in K562 and from 0.45μM to 0.03 in KCL-22 cells. No activity of either compound was observed in the bcr-abl negative HL-60 and KG-1 cells.In primary cells from imatinib-resistant pts, a decrease of the IC50 in imatinib versus AMN107 treated peripheral blood cells from 0.75μM to 0.1μM and from 4 to 0.4μM was detected. In addition, in primary cells from one newly diagnozed CML patient the IC50 of AMN107 (2.5μM) was reduced when compared to imatinib (5μM).Immunoblotting showed that in LAMA84 cells a concentration of 0.01μM AMN107 completely inhibited the tyrosine kinase activity as detected by use of an anti-phosphotyrosine antibody in contrast to almost 5μM in imatinib treated samples.Further, induction of apoptosis was detected using annexin V and propidium iodide by double fluorescence. After 48 hours of incubation with either 0.25 μM imatinib or 0.005 μM AMN107 induction of early apoptosis was detected in 8.8% of imatinib treated and 26% of AMN107 treated cells. Finally, HPLC analysis in HL-60 cells showed increased uptake by 1,5 fold for AMN107 when compared to imatinib. In addition, in MDR1 over-expressing CCRF cells co-culture with either AMN107 or imatinib revealed elevated AMN107 levels (3.7 fold) indicating that this substance is less susceptible to MDR1 driven resistance than imatinib.Conclusions: 1. AMN107 showed elevated activity when compared to imatinib in bcr-abl + cell lines and primary cells derived from imatinib resistant leukemic pts. 2. Complete inhibition of the bcr-abl tyrosine kinase activity and induction of apoptosis was achieved at lower concentrations in AMN107 treated samples when compared to imatinib. 3. Preliminary data indicate favourable cellular uptake of AMN107 when compared to imatinib. 4. AMN107 may be useful in the treatment of bcr-abl + leukemic pts.

Sole BCR-ABL inhibition is insufficient to eliminate all myeloproliferative disorder cell populations

Protein kinase inhibitors can be effective in treating selected cancers, but most suppress several kinases. Imatinib mesylate has been useful in the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia and B cell acute lymphoblastic leukemia through the inhibition of BCR-ABL tyrosine kinase activity. Imatinib mesylate has also been shown to inhibit KIT, ARG, and platelet-derived growth factor receptors alpha and beta, and potentially other tyrosine kinases. We have produced a mutant allele of BCR-ABL (T315A) that is uniquely inhibitable by the small molecule 4-amino-1-tert-butyl-3-(1-naphthyl)pyrazolo[3,4-d]pyrimidine and used it to demonstrate that sole suppression of BCR-ABL activity was insufficient to eliminate BCR-ABL(+) KIT(+)-expressing immature murine myeloid leukemic cells. In contrast, imatinib mesylate effectively eliminated BCR-ABL(+) KIT(+)-expressing leukemic cells. In the cellular context of mature myeloid cells and Pro/Pre B cells that do not express KIT, monospecific BCR-ABL inhibition was quantitatively as effective as imatinib mesylate in suppressing cell growth and inducing apoptosis. These results suggest that the therapeutic effectiveness of small molecule drugs such as imatinib mesylate could be due to the inhibitor's ability to suppress protein kinases in addition to the dominant target.

Tyrosine kinase inhibitor AG1024 exerts antileukaemic effects on STI571-resistant Bcr-Abl expressing cells and decreases AKT phosphorylation

Chronic myelogenous leukaemia (CML) is a clonal malignancy of the pluripotent haematopoietic stem cell, characterised by an uncontrolled proliferation and expansion of myeloid progenitors expressing a fusion oncogene, BCR-ABL, the molecular counterpart of the Ph1 chromosome. The tyrosine kinase (TK) activity of BCR-ABL is known to activate several major signalling pathways in malignant cells, including Ras, JAK/STAT and PI3K/Akt with evidence of proteasome-mediated degradation of other targets such as the DNA repair protein DNA-PKcs and cyclin-dependent kinases inhibitor p27. Targeting these abnormalities by blocking TK of BCR-ABL with STI571 provided a promising approach for the therapy of CML. The recent development of resistance to STI571 illustrates, however, that the use of other TK inhibitors could be of major interest for therapeutic purposes. To this end, the TK inhibitor Tyrphostin AG1024 was used to evaluate effect on regulation of BCR-ABL expression, inhibition of cell proliferation and tumour formation in vivo in human and murine BCR-ABL expressing cell lines. Tyrphostin AG1024 was shown to downregulate expression of BCR-ABL and P-Akt, and to upregulate DNA-PKcs expression. In addition, Tyrphostin AG1024 was able to inhibit cell proliferation, and delay tumour growth in vivo. Thus, AG1024 is able to interfere with three major targets of BCR-ABL in leukaemic cells. Interestingly, Tyrphostin AG1024 was also effective against cells resistant to STI571 by distinct mechanisms including Bcr-Abl mutation. Therefore, these data suggest that Tyrphostin AG1024 could represent the basis of a novel therapy for STI571 refractory CML.

Role of the p38 Mitogen-activated Protein Kinase Pathway in the Generation of the Effects of Imatinib Mesylate (STI571) in BCR-ABL-expressing Cells

Imatinib mesylate (STI571), a specific inhibitor of the BCR-ABL tyrosine kinase, exhibits potent antileukemic effects in vitro and in vivo. Despite the well established role of STI571 in the treatment of chronic myelogenous leukemia, the precise mechanisms by which inhibition of BCR-ABL tyrosine kinase activity results in generation of antileukemic responses remain unknown. In the present study we provide evidence that treatment of CML-derived BCR-ABL-expressing leukemia cells with STI571 results in activation of the p38 mitogen-activated protein (MAP) kinase signaling pathway. Our data indicate that STI571 induces phosphorylation of the p38 and activation of its kinase domain, in KT-1 cells and other BCR-ABL-expressing cell lines. We also identify the kinases MAP kinase-activated protein kinase-2 and Msk1 as two downstream effectors of p38, activated during inhibition of BCR-ABL activity by STI571. Importantly, pharmacological inhibition of p38 reverses the growth inhibitory effects of STI571 on primary leukemic colony-forming unit granulocyte/macrophage progenitors from patients with CML. Altogether, our data establish that activation of the p38 MAP kinase signaling cascade plays an important role in the generation of the effects of STI571 on BCR-ABL-expressing cells. They also suggest that, in addition to activation of mitogenic pathways, BCR-ABL promotes leukemogenesis by suppressing the function of growth inhibitory signaling cascades.

Imatinib mesylate (gleevec; STI571) monotherapy is ineffective in suppressing human anaplastic thyroid carcinoma cell growth in vitro.

Imatinib mesylate is remarkably effective in treating chronic myeloid leukemia and metastatic gastrointestinal stromal tumors. Meanwhile, anaplastic thyroid carcinoma (ATC) remains a fatal malignancy for which there are currently no effective curative interventions. In chronic myeloid leukemia and gastrointestinal stromal tumors, imatinib inhibits the constitutive tyrosine kinase activity of BCR-ABL and c-KIT, respectively. Reports suggest that imatinib may also be effective against ABL and platelet-derived growth factor receptor kinase-dependent pathological conditions. These mechanisms provide a wide scope of possible clinical applications for the drug. Potentially, diseases instigated by constitutive kinase activity that can be inhibited with imatinib should be treatable with this drug. We evaluated the effects of imatinib on the viability, cycling, and tyrosine phosphorylation of ATC cells in vitro. Our data indicate that imatinib has negligible antineoplastic activity against ATC cell lines within established therapeutically useful concentrations. No constitutive kinase activity was detected in these cell lines that could be exploited as a therapeutic target by imatinib. We conclude that imatinib mesylate monotherapy would not be effective in ATC patients. Current preclinical data do not warrant future clinical studies of imatinib monotherapy for ATC.

Mitochondrial dysfunction as an early event in the process of apoptosis induced by woodfordin I in human leukemia K562 cells

Tannins are a group of widely distributed plant polyphenols, some of which are beneficial to health because of their chemopreventive activities. In the present study, we investigated the effects and action mechanisms of woodfordin I, a macrocyclic ellagitannin dimer, on human chronic myelogenous leukemia (CML) K562 cells. The results showed that woodfordin I was able to suppress the proliferation and induce apoptosis in K562 cells. Apoptosis was evaluated by cytomorphology, internucleosomal DNA fragmentation, and externalization of phosphatidylserine. Woodfordin I treatment caused a rapid and sustained loss of mitochondrial transmembrane potential (MMP), transient generation of reactive oxygen species (ROS), transient elevation of intracellular Ca 2+ concentration, and cytosolic accumulation of cytochrome c. The activation of caspase-9 and 3, but not caspase-8, was also demonstrated, indicating that the apoptotic signaling triggered by woodfordin I was mediated through the intrinsic mitochondria-dependent pathway. Western blot and immunofluorescence analysis revealed that the anti-apoptotic Bcl-2 and Bcl-x L levels were downregulated, together with the pro-apoptotic Bax protein. Significantly, woodfordin I-induced apoptosis was associated with a decline in the levels of c-Abl, Bcr-Abl, and cellular protein tyrosine phosphorylation. Considering the consequence of all the events in the process of woodfordin I-induced apoptosis, the mitochondrial dysfunction is directly responsible for the pro-apoptotic effects on K562 cells. Furthermore, because CML is a malignancy of pleuripotent hematopoietic cells caused by the dysregulated tyrosine kinase activity of Bcr-Abl, these findings suggest that woodfordin I may be a potential lead compound against CML.

C-CBL is not required for leukemia induction by Bcr-Abl in mice

Bcr-Abl tyrosine kinase activity is essential for the pathogenesis of chronic myeloid leukemia (CML). A number of Bcr-Abl substrates have been identified, but it is not clear which of these substrates are required for Bcr-Abl to transform cells. The multifunctional protein c-Cbl is one of the most prominently tyrosine-phosphorylated proteins in Bcr-Abl-expressing cells. Using cell lines and mice with homozygous disruption of the c-CBL locus, we investigated the role of this protein for Bcr-Abl-driven transformation. We find that although c-Cbl(-/-) fibroblast cell lines show a deficit in Bcr-Abl transformation compared to wild-type (Wt) cells, this deficit was less pronounced in c-Cbl(-/-) B cells derived from murine bone marrow. Most importantly, in a transplantation model of CML, Bcr-Abl was capable of inducing fatal leukemia in mice in the absence of c-Cbl protein. Our results indicate that c-Cbl is dispensable for Bcr-Abl-induced leukemogenesis in mice.

Un nouveau traitement pour la leucémie myéloïde chronique : L'imatinib (glivec ®) implications pour l'approche diagnostique et le suivi biologique de la maladie

Imatinib-Gleevec ® is a new therapeutic approach of chronic myelogenous leukemia (CML). Its action is directed against the BCR-ABL transcript wich characterizes the disease. Imatinib-Gleevec is an inhibitor of the tyrosine kinase activity of BCR-ABL. The results of phase II and III trials have surpassed all the predictions. In the phase III study, at 18 months, 74 % of Imatinib treated patients were in complete cytogenetic remission versus 8 % of interféron + aracytine treated patients. However the long term results with imatinib and its ability to cure CML, alone or in association with other therapies, remain to be determined. With this new treatment, the clinical pathologist has a pivotal role for an early diagnosis as well as for the follow-up of the treatment.

Innovations in the management of leukemia: role of biologic therapies.

Chronic myelogenous leukemia (CML) results from clonal proliferation of malignant hematologic progenitor cells and is responsible for 7% to 15% of all adult leukemias. The hallmark of CML is a genetic translocation between chromosomes 9 and 22 (the Philadelphia chromosome), which creates the abnormal bcr-abl gene and a continuously activated Bcr-Abl protein. Bcr-Abl tyrosine kinase activity leads to signal transduction and cell growth. Traditional therapies for CML include allogeneic stem cell transplantation, interferon alfa, and oral chemotherapeutic agents. However, because Bcr-Abl is the causative abnormality in CML, it represents an ideal target for rational biologic therapy. Imatinib mesylate is an orally available tyrosine kinase inhibitor that specifically blocks Bcr-Abl function. Clinical trials have demonstrated that imatinib mesylate produces rapid responses in patients with all stages of CML, including those who were resistant to interferon alfa therapy or intolerant of it. When imatinib mesylate therapy was initiated early in the course of CML, there was a complete hematologic response in 98% of the patients and a complete cytogenetic response in 72% of the patients. Although long-term safety and survival data are not yet available, imatinib mesylate is a promising new treatment option for CML that targets the molecular cause of the disease.

Basic science going clinical: molecularly targeted therapy of chronic myelogenous leukemia.

Imatinib (STI571), a 2-phenylaminopyrimidine, specifically inhibits the tyrosine kinase activity of Abl, Kit, and platelet-derived growth factor receptor. Clinical trials in chronic myelogenous leukemia (CML), characterized by the constitutively active Bcr-Abl tyrosine kinase, and gastrointestinal stromal tumors, characterized by activating mutations of Kit, have shown excellent results. This success is proof of principle for the concept of molecularly targeted therapy: rational treatment based on the recognition of the causal lesion responsible for malignant growth. In this manuscript, the preclinical and clinical development of imatinib for the treatment of CML will be reviewed. Room will be given to problems and challenges that may be typical of molecularly targeted therapy in general, such as the emergence of resistance as a result of point mutations. Last, the question will be addressed, why imatinib is so successful, and whether its success might be reproducible in other malignant conditions.

IL-3 receptor signaling is dispensable for BCR-ABL-induced myeloproliferative disease

BCR-ABL expression led to a dramatic up-regulation of the IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptor beta common (IL-3Rbetac) and IL-3 receptor beta (IL-3Rbeta) chains in murine embryonic stem cell-derived hematopoietic cells coincident with an expansion of multipotent progenitors and myeloid elements. This up-regulation required BCR-ABL tyrosine kinase activity and led to IL-3Rbetac/beta chain tyrosine phosphorylation in the absence of detectable IL-3 production. These results suggested that cytokine-independent IL-3 receptor activation could be a dominant signaling component in BCR-ABL-induced leukemogenesis. To unambiguously define the significance of IL-3 receptor-dependent signaling in BCR-ABL-induced leukemogenesis, BCR-ABL-transduced bone marrow cells deficient in either IL-3Rbetac chain or both IL-3Rbetac/beta chain expression were examined for their ability in generating myeloproliferative disease (MPD). These BCR-ABL-expressing knockout cells were capable of generating MPD similar to control cells, demonstrating that IL-3 receptor activation is not essential for BCR-ABL-induced MPD. However, the IL-3Rbetac/beta chain could act as a cofactor in BCR-ABL-induced leukemogenesis by activation of its many known oncogenic signaling pathways.