BCR-ABL Expressing Cell Lines Research Articles

Fingolimod (FTY720) Inhibits BCR-ABL Signaling Allosterically by Binding to the Myristate Binding Site

Abstract 2746 Background:The tyrosine kinase (TK) activity of the ABL protein is normally tightly regulated, with the N-terminal cap (NCap) region (≈ 80 amino-acid residues) of the SH3 domain playing an important role. One regulatory mechanism involves the NCap Gly-2 residue being myristoylated and then interacting with a myristate binding site within the SH1 catalytic domain. Chronic myeloid leukemia (CML) results from a chromosome translocation leading to expression of the chimeric BCR-ABL oncoprotein, which lacks the NCap and has constitutively active TK activity. Although drugs that inhibit the TK activity of the BCR-ABL oncoprotein via an ATP-competitive mechanism are effective in the treatment of chronic myeloid leukemia (CML), some patients relapse due to the emergence of drug-resistant clones, in which mutations in the SH1 domain compromise inhibitor binding. Recently, we have shown that compounds that bind to the SH1 myristate pocket can also inhibit the TK activity of BCR-ABL, and that these maintain activity against mutant enzymes (Nature 2010;263:501). FTY720 is a drug approved for the treatment of multiple sclerosis which, upon phosphorylation, targets sphingosine-1-phosphate receptors leading to immunomodulatory activity. FTY720 also activates the tumour suppressor protein phosphatase 2A, that is inactivated in CML and upon reactivation impairs BCR-ABL expression and function, leading to growth suppression, enhanced apoptosis, impaired clonogenicity, and decreased in vivo leukemogenesis of imatinib-sensitive and -resistant BCR-ABL expressing cell lines and primary myeloid BP-CML cells (Br J Cancer 2006;95:775). Based upon structural insight it was postulated that FTY720 might bind to the ABL myristate site, and here we report on both the binding and allosteric inhibition of ABL TK activity. Methods and Results:In order to evaluate whether FTY720 could interact with ABL, nuclear magnetic resonance (NMR) studies were employed: In a conformational assay (JACS 2010;132:7043), FTY720 induced bending of the SH1 C-terminal helix I, that is a prerequisite for inhibitory activity of allosteric BCR-ABL ligands, and showed a binding affinity, Kd of 3 μM. The effects of FTY720 and it’s phosphorylated analogue on the kinase activity of ABL64–515 (containing SH3SH2SH1 domains, lacking the C-terminal domain encoded by exon 11) and ABL229–515 (SH1 domain only) were determined using radiometic filter-binding assays using Poly-(AlaGluLysTyr) as protein substrate (Biochim Biophys Acta 2010;1804:454). FTY720 dose-dependently inhibited the transphosphorylation activity of ABL64–515 with an IC50 value of 1.4 ± 1.1 μM, but did not effect the catalytic activity of ABL229–515 at concentrations < 25 μM; furthermore phosphorylated-FTY720 was inactive. However, in pharmacological assays FTY720 only weakly inhibited the BCR-ABL dependent proliferation of either murine 32D or Ba/F3 cells (GI50 > 3 μM) and had, in comparison to nilotinib, modest dose-dependent effects in a murine leukemia model (52% reduction in growth compared to vehicle-treated controls at 10 mg/kg p.o. q24h; nilotinib gave 89% reduction at 20 mg/kg q24h). Discussion:Biochemical and biophysical data confirm that FTY720 is an allosteric inhibitor of the TK activity of ABL. Although the extent of this effect is insufficient to produce substantial efficacy in BCR-ABL transfected cells, the findings are of mechanistic interest since they probably contribute to the previously published effects of FTY720 in CML models and provide a new structural lead for allosteric inhibitors of ABL. Disclosures:Manley:Novartis Pharma AG: Employment. Cowan-Jacob:Novartis Pharma AG: Employment. Cozens:Novartis Pharma AG: Employment. Fendrich:Novartis Pharma AG: Employment. Jahnke:Novartis Pharma AG: Employment. Roesel:Novartis Pharma AG: Employment. Fabbro:Novartis Pharma AG: Employment.

Role of BCR-ABL-Y177-mediated p27kip1 phosphorylation and cytoplasmic localization in enhanced proliferation of chronic myeloid leukemia progenitors

In chronic myelogenous leukemia (CML) hematopoietic stem cell transformation leads to increased proliferation of malignant myeloid progenitors. The cyclin-dependent kinase inhibitor p27kip1 (p27) is a critical negative regulator of hematopoietic progenitor proliferation and pool size that is deregulated in BCR-ABL expressing cell lines. However, cell-context specific regulation of p27 in primary human CML progenitors and its contribution to CML progenitor expansion remain unclear. Here we investigated p27 regulation and function in (1) CD34+ cells from CML patients and (2) human CD34+ cells ectopically expressing the BCR-ABL gene following retrovirus transduction. We found that p27 levels are increased in CML CD34+ cells related to a BCR-ABL dependent increase in p27 protein translation. However p27 was relocated to the cytoplasm in CML progenitors and nuclear p27 levels were reduced, allowing increased cell cycling and expansion in culture. Cytoplasmic relocation of p27 in CML progenitors was related to signaling through BCR-ABL Y177, activation of the AKT kinase and phosphorylation of p27 on Thr-157 (T157). Expression of a mutant p27 that cannot be phosphorylated on T157 significant inhibited CML progenitor proliferation. These studies demonstrate the importance of BCR-ABL-Y177-AKT mediated p27 phosphorylation in altered p27 localization and enhanced proliferation and expansion of primary CML progenitors.

Defining Extrinsic Survival Pathways in CML Progenitor Cells.

Abstract 941Defining extrinsic survival pathways in CML progenitor cellsRyan MacKenzie1, Jennifer Snead1, Jonathan VanDyke1, Brian Druker1,2, Michael Deininger11Center for Hematologic Malignancies, Oregon Health & Science University Cancer Institute, 2Howard Hughes Medical Institute, 3181 S.W. Sam Jackson Park Rd., Portland, Oregon 97239-3098, USA Background:The stem cell niche is thought to provide a protective microenvironment that allows for the persistence of malignant progenitors in various types of leukemia treated with chemotherapy. Tyrosine kinase inhibitor (TKI) treatment of chronic myeloid leukemia (CML) does not completely eliminate residual BCR/ABL positive progenitors, despite complete cytogenetic responses in the majority of patients, a phenomenon referred to as disease persistence. We hypothesize that niche factors may contribute to CML progenitor survival in the presence of imatinib and that the identification of defined survival factors may lead to therapeutic approaches to eliminate residual disease. Approach and results:In an initial series of experiments CD34+ CML progenitor cells were cultured in (a) serum free media supplemented with low concentrations of cytokines (=control), (b) in direct contact with M210B4 murine stroma or (c) separated from M210B4 murine stroma through a transwell membrane, in the presence or absence of TKIs (dasatinib, imatinib). After 96h, cells were plated in semisolid media and CFU-GM scored. Murine stroma consistently increased CFU-GM survival 4-5-fold compared to controls, with most of the effect attributable to soluble factors. Similar observations were made with human stroma (HS-5), although direct contact accounted for approximately 40% of the protective effect. Since integrin-mediated adhesion has been shown to protect CML cells lines from imatinib and chemotherapy, we hypothesized that direct contact protection of CML CD34+ cells may be related to integrin engagement. To test this hypothesis we cultured CML CD34+ cells on plates coated or not with fibronectin and in the presence or absence of integrin activating (B44) or blocking antibodies (6S6), with imatinib added at 1.5 and 5 uM or dasatinib added at 5 or 50 nM. However, in several experiments (N=3), we found that integrin-mediated adhesion to fibronectin failed to promote CFU-GM survival in the presence of TKIs. To identify soluble factors responsible for progenitor cell survival in the presence of TKIs we measured cytokine and chemokine concentrations in HS-5 stroma conditioned media (CM) and found measurable levels of several cytokines with JAK2-dependent signal transduction, including IL3, IL6, GM-CSF and G-CSF. Based on this we asked whether a JAK2 inhibitor (CYT387) would synergize with imatinib to inhibit CML CFU-GM survival. Thus, CD34+ cells were cultured for 96h with imatinib and CYT387 HS-5 stroma cells. Imatinib (5uM) reduced CFU-GM survival by 79.4%. Addition of 5 uM of CYT387, a JAK2 inhibitor (IC50 for growth inhibition of BAF3 cells expressing JAK2V617F: 1.5uM) reduced CFU-GM survival by 92.9%. Combination of imatinib with graded concentrations of CYT387 led to a further reduction of CFU-GM in a dose-dependent manner (Table 1).Table 1:Percent of untreated CFU-GM numbers derived from CD34+ CML cells after a 4-day culture in various conditions.imatinib0uM0uM5uM5uM5uM5uM4 Day Culture ConditionsCYT3870uM5uM0uM0.5uM2.5uM5uMOn HS5 stroma100.07.120.63.52.91.0 Conclusions:(a) Contact to and soluble factors produced by stromal cells appear to protect CD34+ CML progenitor cells in the presence of TKIs. (b) We find no evidence that integrin-mediated adhesion to fibronectin is involved in the protection of primary cells (unlike BCR-ABL expressing cell lines). (c) Combination of a JAK2 inhibitor with imatinib synergistically reduces colony formation, suggesting that this combination may be effective in targeting residual leukemia cells in vivo. Disclosures: Deininger:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Calistoga: Research Funding; Genzyme: Research Funding.

Potentiation of antileukemic therapies by the dual PI3K/PDK-1 inhibitor, BAG956: effects on BCR-ABL– and mutant FLT3-expressing cells

AbstractMediators of PI3K/AKT signaling have been implicated in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). Studies have shown that inhibitors of PI3K/AKT signaling, such as wortmannin and LY294002, are able to inhibit CML and AML cell proliferation and synergize with targeted tyrosine kinase inhi-bitors. We investigated the ability of BAG956, a dual PI3K/PDK-1 inhibitor, to be used in combination with inhibitors of BCR-ABL and mutant FLT3, as well as with the mTOR inhibitor, rapamycin, and the rapamycin derivative, RAD001. BAG956 was shown to block AKT phosphorylation induced by BCR-ABL–, and induce apoptosis of BCR-ABL–expressing cell lines and patient bone marrow cells at concentrations that also inhibit PI3K signaling. Enhancement of the inhibitory effects of the tyrosine kinase inhibitors, imatinib and nilotinib, by BAG956 was demonstrated against BCR-ABL expressing cells both in vitro and in vivo. We have also shown that BAG956 is effective against mutant FLT3-expressing cell lines and AML patient bone marrow cells. Enhancement of the inhibitory effects of the tyrosine kinase inhibitor, PKC412, by BAG956 was demonstrated against mutant FLT3-expressing cells. Finally, BAG956 and rapamycin/RAD001 were shown to combine in a nonantagonistic fashion against BCR-ABL– and mutant FLT3-expressing cells both in vitro and in vivo.

Enhanced Phosphorylation and Altered Localization Lead to Impairment of p27kip Activity in CML Progenitor Cells Despite Enhanced Protein Translation and Expression.

The cyclin-dependent kinase inhibitor p27kip is a key regulator of hematopoietic progenitor proliferation and pool size. The activity of p27 can be regulated by modulation of its expression and localization in different cellular compartments. The levels of p27 protein expression are reported to be reduced in BCR-ABL expressing cell lines. In contrast p27 levels have been reported to be elevated in BCR/ABL expressing CML progenitor cells. However, CML progenitors paradoxically demonstrate enhanced proliferation despite having elevated levels of p27. Therefore the regulation of p27 protein expression and activity in primary CML progenitor cells is not well understood and requires further investigation. We have therefore performed a careful assessment of p27 expression, phosphorylation and localization in CD34+ cells from CML patients as well as in cord blood CD34+ cells transduced with retroviral vectors to ectopically express the BCR-ABL gene. We observed that p27 protein levels were markedly increased in both primary CML CD34+ cells and BCR/ABL transduced cells. CML CD34+ cells and BCR/ABL transduced CD34+ cells also demonstrated increased phosphorylation of p27 on T157 (an Akt-dependent phosphorylation site) and T187 (a cdk2-dependent site) on western blotting as well as increased tyrosine phosphorylation of p27 in immunoprecipitation studies. Immunofluorescence microscopy analysis of cells labeled with anti-p27 antibodies demonstrated primarily nuclear localization of p27 in normal CD34+ cells, but markedly reduced nuclear and increased cytoplasmic localization of p27 in CML CD34+ cells and BCR/ABL transduced CD34+ cells. Cdk2 activity was maintained, cdk4 activity was increased and phosphorylated RB levels were increased in BCR-ABL expressing hematopoietic cells, indicating CDKI activity was reduced in these cells despite increased total p27 levels. Consistent with this BCR-ABL expressing CD34+ cells demonstrated enhanced cell cycling compared to controls. Mutation of the Y177 residue in BCR-ABL (BCR-ABL-Y177F), which abrogates Grb2 binding and reduces Ras and Akt activation by BCR-ABL, reversed BCR-ABL induced abnormalities in p27 expression, phosphorylation and localization. Treatment of BCR-ABL transduced CD34+ cells with the PI-3K inhibitor LY294002 resulted in reduced phosphorylation of p27 on T157 but not T187, and restored nuclear localization of p27, further indicating an important role for AKT signaling in abnormal cytoplasmic localization of p27. Quantitative reverse transcription PCR analysis indicated that p27 mRNA levels were similar in BCR/ABL expressing and control CD34+ cells, suggesting that p27 expression is regulated mainly at the posttranscriptional level. Metabolic labeling of cells with S35-methionine showed that p27 translation was increased in BCR/ABL expressing cells, explaining the observed increase in total p27 protein levels. We conclude that p27 CDKI activity is reduced in BCR-ABL expressing progenitors as a result of altered p27 phosphorylation at important regulatory sites and a shift in cellular localization of p27 from the nucleus to the cytoplasm. The enhanced level of total p27 in primary CML progenitors reflects enhanced protein translation, which may be a compensatory response to reduced p27 CDKI activity. These results provide important insights into p27 deregulation in CML progenitors and its potential role in the expansion of the hematopoietic progenitor pool size in CML.

A Phase I Study of INNO-406 in Patients with Advanced Philadelphia (Ph+) Chromosome-Positive Leukemias Who Are Resistant or Intolerant to Imatinib and Second Generation Tyrosine Kinase Inhibitors.

Background: INNO-406 is an orally available, dual Abl/Lyn kinase inhibitor that is up to 55-times more potent than imatinib in Bcr-Abl expressing cell lines. Numerous Bcr-Abl mutant proteins are sensitive to INNO-406 in vitro, including the F317L mutant. Unlike other second generation tyrosine kinase inhibitors (TKIs), INNO-406 demonstrates specific Lyn kinase activity with no or limited activity against other Src-family member kinases.Methods: In this phase I dose finding study, patients (pts) with imatinib-resistant or -intolerant Philadelphia (Ph+) chromosome-positive leukemias were eligible for treatment with INNO-406 orally at doses ranging from 30 mg once daily (qd) through 480 mg twice daily (bid).Results: INNO-406 was administered to 41 pts (23M, 18F); median age 61 yrs (range, 20–76). Median duration of CML was 6 yrs (range, 0.1–23); median time on imatinib was 0.8 yrs (range, 0.1–6.5). Pts had CML in chronic phase (CP, n=21), accelerated phase (AP, n=7), or blast phase (BP, n=6), or Ph+ ALL (n=7). Previous treatment included nilotinib (n=9), dasatinib (n=13), and dasatinib/nilotinib (n=10). Pts have been treated with INNO-406 for a median of 42 days (range, 7–240+). Common mutations on study entry included Y253H (n=4), F311L (n=3), F317L (n=2), and T315I (n=2). Currently, 14 pts remain on study; 22 pts discontinued due to disease progression, 4 pts discontinued to pursue other treatment options, and 1 pt discontinued due to toxicity. Hematologic and cytogenetic responses have been reported. 2 of 7 CP pts who had failed only imatinib demonstrated a complete cytogenetic response, including 1 major molecular response. An additional pt had a minor cytogenetic response after 4 weeks of therapy before going off study to pursue an alternate treatment option. Among pts in CP who had failed more than one TKI, a complete cytogenetic response was reported in 1 pt who was intolerant to both imatinib (lung/skin toxicity) and dasatinib (pleural effusion). All 7 AP pts on study had failed multiple TKIs. In this population, 2 pts experienced complete hematologic responses. Additionally, in 1 pt, a reversion of the Y253H mutation was demonstrated, and 1 pt has experienced a disappearance of the F317L mutation. Related adverse events include reversible grade 2/3 elevation in the activity of transaminases. A DLT was reported in 1 pt in the 480 mg bid cohort (intrahepatic cholestasis and renal failure secondary to tumor lysis syndrome).Conclusions: INNO-406 is well tolerated in pts with clinical activity demonstrated across a range of dosing. Responses occur even in the setting of a heavily pretreated population thus making INNO-406 a viable option for CML therapy. The recommended phase II dose is anticipated to be 240 mg bid. Pivotal phase II studies are planned for late 2007.

Synergistic Activity of the Novel Src/Abl Inhibitor Bosutinib in Combination with Imatinib.

The oncogenic fusion protein Bcr-Abl is the underlying cause of Chronic Myeloid Leukemia (CML), and it is present in up to 35% of Acute Lymphoblastic Leukemia (ALL). The discovery of a selective Abl inhibitor, Imatinib mesylate, has revolutionized the treatment of CML. Recently, several new inhibitors have been developed with the aim of increasing both potency and selectivity against Abl. Bosutinib (SKI-606, Wyeth) is a dual Src/Abl inhibitor that showed an in vitro activity in the low nanomolar range on several BCR-ABL positive cells and it is, at present, in phase II clinical trials. Bosutinib is devoid of activity against some known “off-target” kinases blocked by imatinib, such as PDGFR and c-Kit. In addition structural and modelling data attribute to Bosutinib the ability to bind Bcr-Abl in the intermediate/active conformation, while Imatinib is able to bind only the inactive conformation of Bcr-Abl. In this study we analyzed in vitro the combination of Imatinib and Bosutinib in Bcr-Abl expressing cell lines to evaluate the possibility to decrease dosage of both drugs, increasing or maintaining the same efficacy but avoiding toxic effects. Combination effects were evaluated according to the method of Chou and Talalay, in which the combination index (CI) value is calculated for a combination of two drugs and allows the quantification of synergism: CI <1, =1 or >1 indicate synergistic, additive or antagonistic interactions, respectively. Proliferation assays on a panel of Imatinib-sensitive and Imatinib-resistant BCR-ABL positive cell lines were performed. Cells were treated with Imatinib and Bosutinib as single agents or in three ratio combinations (1:3, 1:10, 1:33 in favour to Imatinib) across a wide range of concentrations. Combination indexes (CI) calculated at IC50, IC75 and IC90 for K562 cells (Imatinib-sensitive), suggest a synergistic to very strong synergistic effect (CI= 0.01-0.53). Similarly, in KCl22, KU812 and Lama84 cells (Imatinib-sensitive) moderate to strong synergistic effects were observed. A slight to moderate synergism was also obtained in three Imatinib-resistant cell lines tested: Lama84R (CI=0.63-0.88), K562R (CI=0.63-0.82) and KCL22R (CI=0.62-0.92). Western blot analysis of the tyrosine phosphorylation status of K562S cells treated with a mixture of 100nM Imatinib and 10nM Bosutinib revealed a substantially more pronounced inhibition compared with either 100nM Imatinib or 10nM Bosutinib alone. The effect of the combination was also assessed in murine Ba/F3 cells transfected with either wild type (WT) or mutated forms of BCR-ABL. Parental Ba/F3 cells were not affected by the presence of both drugs, while in Ba/F3 BCR-ABL WT the CI ranged from 0.49 to 0.85, indicating moderate synergism. The combination of Imatinib and Bosutinib inhibited the growth of Ba/F3 BCR-ABL Y253F with a slight synergism (CI 0.77-0.87). No synergistic effect was observed on Ba/F3 BCR-ABL E255K and on the highly resistant T315I mutant. Fresh leukemic cells obtained from three CML patients were also studied. In these samples synergistic effects between Bosutinib and Imatinib were confirmed (CI=0.52, 0.73, 0.62). The different binding modes of Imatinib and Bosutinib may justify the synergistic effect observed in the CML lines. This results support a possible therapeutic advantage for the combination of Bosutinib and Imatinib against Philadelphia positive leukemias.

Effective Induction of Apoptosis in Chronic Myeloid Leukemia CD34+ Cells by the Histone Deacetylase Inhibitor LAQ824 in Combination with Imatinib.

Imatinib mesylate (IM) is highly effective in the treatment of chronic myeloid leukemia (CML). However IM does not eliminate quiescent, primitive CML hematopoietic cells, which persist through treatment and remain a potential source of relapse. Therefore additional interventions are required to more effectively target primitive CML cells. The hydroxamic acid analogue histone deacetylase (HDAC) inhibitors, LAQ824, and LBH589, are reported to induce apoptosis and inhibit cell cycle in CML cell lines and blast crisis (BC) CML cells. Although most pro-apoptotic agents preferentially target dividing cells, HDAC inhibitors are a rare class of drugs reported to induce apoptosis in non-dividing cells. We were therefore interested in investigating whether the HDAC inhibitor LAQ824 (LAQ) could induce apoptosis in CD34+ cells from chronic-phase (CP) CML patients. CML and normal CD34+ cells were cultured with LAQ (10–100nM) alone and in combination with IM (1μM) for 96 hours in growth factor-supplemented medium. Treatment with LAQ (25–100 nM) effectively enhanced Histone H3 acetylation in CML and normal CD34+ cells as detected by Western blotting with an anti-Histone H3 (Lys9/Lys14) antibody. Although LAQ treatment by itself induced significantly less apoptosis in CML compared to normal CD34+ cells, LAQ in combination with IM was highly effective in inducing apoptosis in CML CD34+ cells (11+/−3%, apoptosis with IM alone, 18+/−3% with IM+10 nM LAQ, 22+/−2% with IM+25 nM LAQ, and 32+/−3% with IM+50 nM LAQ824, n=5). Significantly higher levels of apoptosis were seen in CML compared with normal CD34+ cells at lower doses of LAQ (p=0.03 at 10 nM). LAQ combined with IM also resulted in enhanced apoptosis of non-dividing CML CD34+ cells, identified using CFSE labeling, with 43% apoptosis observed in cells treated with 50 nM LAQ [n=5]. Combined LAQ and IM treatment also resulted in significant inhibition of CML progenitor proliferation measured in CFSE assays [n=4] and in the number of CML progenitors measured in methylcellulose progenitor assays [n=5]. In contrast to what has been described for BCR-ABL expressing cell lines and BC CML cells, LAQ treatment did not result in significant change in BCR-ABL levels in CML CD34+ cells on Western blotting. Significant down-regulation of MCL-1, a critical apoptosis regulator in early hematopoietic cells, was observed on Western blotting of CML cells treated with the LAQ and IM combination [n=5]. Levels of other anti-apoptotic proteins BCL-xL and BCL-2 were not affected. MCL-1 inhibition was not observed in normal CD34+ cells [n=4]. In contrast, normal cells treated with LAQ or LAQ and IM demonstrated enhanced levels of the pro-apoptotic protein Bim, but Bim levels were not increased in CML CD34+ cells. Q-PCR analysis revealed changes in MCL-1 and BIM mRNA levels in CML and normal CD34+ cells with LAQ and IM treatment that were consistent with the results of Western blotting. We conclude that treatment with the HDAC inhibitor LAQ in combination with imatinib results in significantly enhanced apoptosis of CML CD34+ cells, including non-dividing cells, compared with IM alone. Different mechanisms appear to be responsible for HDAC induced apoptosis in CML and normal CD34+ cells. Our results provide a strong rationale for future clinical application of HDAC inhibitors to enhance elimination of CML progenitors in combination with BCR-ABL tyrosine kinase inhibitors.

Imatinib and Nilotinib induce apoptosis of chronic myeloid leukemia cells through a Bim-dependant pathway modulated by cytokines

It is an important challenge to better understand the mechanisms of tyrosine kinase inhibitors-induced apoptosis in CML cells. Thus, we have investigated how this apoptosis can be modulated by extracellular factors. Apoptosis induced by imatinib and nilotinib was determined in BCR-ABL expressing cell lines and primary CML CD34+ cells. Both molecules induced apoptosis of BCR-ABL expressing cells. This apoptosis was inhibited by protein synthesis inhibition in both K562 and CML CD34+ cells. In K562, 80% inhibition of the BCR-ABL auto-phosphorylation by either imatinib or nilotinib induced a two fold increase in Bim-EL expression and induction of apoptosis in 48h. Bim accumulation preceded apoptosis induction and was completely abolished by depletion in Bim using shRNA. However, the anti-proliferative effect of imatinib was preserved in Bim-depleted cells. When K562 cells were cultured in a cytokine containing medium, the pro-apoptotic effect of nilotinib was decreased by 68% and this was related to a decrease in Bim-EL de-phosphorylation and accumulation. Similarly, the presence of a combination of cytokines inhibited 88% of NIL- and 39% of IMA-induced apoptosis in primary CML CD34+ cells. In conclusion, both nilotinib and imatinib induce apoptosis through Bim accumulation independently of cell cycle arrest. However, the pro-apoptotic effect of both molecules can be attenuated by the presence of cytokines and growth factors, particularly concerning nilotinib. Thus BCR-ABL inhibition restores the cytokine dependence but is not sufficient to induce apoptosis when other signaling pathways are activated.

The Effect of Imatinib Mesylate on the Signal Transduction Cascade Leading to down Regulation of Telomerase Activity in K562 Cells.

Imatinib mesylate (IM) is a tyrosine kinase inhibitor selective for Bcr-Abl, c-Kit, and platelet-derived growth factor receptor kinases. IM inhibits kinase activity through competition for ATP binding. IM is indicated for the treatment of newly diagnosed patients with Philadelphia chromosome positive (BCR-ABL positive) chronic myeloid leukemia (CML) in chronic phase. Recent publications demonstrate that this drug may also target other cellular components. In light of the important role of telomerase in malignant transformation, we evaluated the effect of IM on the signal transduction cascade leading to modulation of telomerase activity in the BCR-ABL positive K562 cell-line. In addition, we studied the effect of IM on telomerase in an IM resistant K562 cell-line (K562res). The K562res cell-line is resistant to the effect of IM on the BCR-ABL protein. However, the effect of IM on telomerase in these cells is unknown. Therefore, using these cells will enable us to study the connection between the signal transduction cascade of BCR-ABL and that of telomerase.IM caused an 80% inhibition of telomerase activity in both K562 and K562res cell-lines. Inhibition of telomerase activity was associated with 50% inhibition of proliferation. Inhibition of telomerase activity was not caused by changes in the transcription level of hTERT (the catalytic subunit of the enzyme). On the other hand, it seemed mainly to be caused by post-translational modifications only in K562 cells namely, a 2-fold dephosphorylation of AKT, known to phosphorylate telomerase. In addition, IM was found to upregulate the expression of PTEN (known to negatively regulate AKT) again only in K562 cells. IM did not affect the levels of phosphorylaetd AKT or PTEN in K562res cells. However, IM caused an approximate 2-fold upregulation of p53 (a transcription factor of PTEN) levels both in K562 and K562res cells.Our results demonstrate the ability of IM to inhibit telomerase activity in BCR-ABL expressing cell lines. Telomerase activity inhibition was demonstrated in K562 cells as well as in K562 cells resistant to high concentrations of IM. Therefore, the inhibitory effect of IM on telomerase activity isn't necessarily mediated through the known tyrosine kinase targets of IM. The effect of IM on the signal transduction cascade leading to the down-regulation of telomerase activity seems to be different in K562 cells and K562res cells. While the signal transduction cascade in K562 cells appears to go through AKT and PTEN, the response to IM in K562res cells seems to be mediated by other yet unknown pathways. Future work is needed in order to clarify possible mechanism(s) by which IM down-regulates telomerase activity in these cells.This study supports previous studies demonstrating telomerase activity as an additional cellular target of IM. In addition, this study shows that cells known to be resistant to IM with regards to its effect on BCR-ABL could still be sensitive to IM treatment regarding other cellular components.

Regulation of pathways downstream of mTOR by imatinib mesylate in CML cells

6595 Background: The differential regulation of signaling pathways downstream of the BCR-ABL tyrosine kinase are being defined in an effort to improve therapeutic strategies for patients with CML. We have previously shown that imatinib mesylate suppressed p70 S6 kinase activity, consistent with inhibition of BCR-ABL-mediated activation of the mTOR/p70 S6 kinase pathway. In this study we examined the effects of imatinib mesylate on other target proteins downstream of mTOR. Methods: BCR-ABL expressing cell lines (K562, KT1 and BV173) were treated with 1μM of imatinib mesylate for 30, 60 and 90 minutes, and then lysed in phosphorylation lysis buffer. Immunoblotting was done using standard enhanced chemiluminescence (ECL) methods. In the experiments in which the pharmacologic inhibitor of mTOR, rapamycin was used, the cells were pretreated for 60 minutes with the inhibitor and subsequently treated with imatinib. Results: The translational repressor 4E-BP1 is constitutively phosphorylated in CML cells. In the presence of imatinib mesylate in concentrations as low as 1μM, the phosphorylation of 4E-BP1 on threonine sites 37 and 46 is decreased. The phosphorylation of eIF-4G on serine site 1108 is also inhibited by imatinib mesylate treatment as early as 60 minutes. The combination of the mTOR inhibitor rapamycin and imatinib mesylate was more potent in suppressing phosphorylation of 4E-BP1 than imatinib alone, suggesting a mechanism for the enhancing effects of rapamycin on imatinib mesylate-dependent inhibition of growth of BCR-ABL cells. Conclusions: Our results further suggest the potential development of future clinical-translational studies utilizing rapamycin in combination with imatinib for the treatment of patients with CML. No significant financial relationships to disclose.

Selective Inhibition of Lysophosphatidic Acid Acyltransferase-β by CT32228 Inhibits MAPK and Enhances the Antileukemic Activity of Imatinib in BCR-ABL Expressing Cell Lines Resistant to Imatinib.

Background: Lysophosphatidic acid acyltransferase-β (LPAAT-β) catalyzes the conversion of lysophosphatidic acid (LPA) to phosphatidic acid (PA), an essential component of the Ras pathway. In vascular smooth muscle cells, specific inhibitors of LPAAT-β interfere with the ras and PI3-kinase dependent signaling pathways thereby inducing growth arrest and apoptosis. We have previously shown that CT32228, a specific inhibitor of LPAAT-β, selectively inhibits the growth of CML progenitor cells in cultures without cytokines [ASH 2003, #2415]. Since Bcr-Abl is usually reactivated in cells resistant to the Abl kinase inhibitor imatinib, and Ras and PI3 kinase are downstream targets of Bcr-Abl, we hypothesized that a specific inhibitor of LPAAT-β may have activity against Bcr-Abl-positive CML with resistance to imatinib.Methods: Antiproliferative activity was assessed using the MTS-proliferation assay. Statistical analysis of combination studies (CT32228 + imatinib) was performed by applying the median effect method. Flow cytometry after staining with propidium iodide (PI) was performed to assess the cell cycle, co-staining with annexin allowed the detection of apoptotic cells. Signaling studies were performed by immunoblotting.Results: CT32228 inhibited the proliferation of Bcr-Abl expressing cell lines (AR230-s, K562, KCL22, Baf/BCR-ABL-s, 32DBCR-ABL, MO7p210) with IC50 concentrations between 23 and 105 nM. Similar IC50 values were obtained in lines expressing imatinib resistant BCR-ABL-mutants (Y253F/M351T/T315I/H396R) or amplified wild-type-BCR-ABL (Baf/BCR-ABL-r1, AR230-r), demonstrating the absence of significant cross-resistance. Combination studies with imatinib demonstrated additive to synergistic antiproliferative activity only in cell lines with residual sensitivity towards imatinib. FACS analysis of K562 cells treated with CT32228 showed accumulation of cells in G2/M as early as 6 h after start of treatment. After 24 h, 50% of cells were in G2/M and apoptosis was demonstrable with annexin/PI staining. Consistent with the G2/M arrest, multinucleated cells were evident microscopically at 24 h. Further analysis of apoptotic signalling pathways revealed activation of caspase-3 and PARP cleavage, while activation of caspase-8 and -9 was not demonstrated. In K562 cells treated with CT32228, Erk1/2 phosphorylation was abrogated, consistent with inhibition of mitogen activated kinase signalling as a result of decreased Ras signalling. In contrast phosphorylation of Akt was unaffected suggesting that inhibition of LPAAT-β does not interfere with the PI3K pathway in BCR-ABL-positive cells.Conclusion: CT32228 exerts antileukemic activity in imatinib-resistant cells without cross-resistance to imatinib and is synergistic in combination with imatinib. Cytotoxicity is mediated by cell cycle arrest in G2/M and induction of apoptosis. Interference with the Ras-pathway through MAPK-inhibition may play a key role in the antileukemic effect of CT32228. This data provides the framework for evaluation of CT32228 and related compounds to improve imatinib-based treatment of CML.

AMN107, a Novel Aminopyrimidine Inhibitor of Bcr-Abl, Has Significant Activity in Imatinib-Resistant Chronic Myeloid Leukemia (CML) or Philadelphia-Chromosome Positive Acute Lymphoid Leukemia (Ph + ALL).

AMN107, a novel aminopyrimidine ATP-competitive inhibitor of Bcr-Abl, is 10-50 fold more potent than imatinib (IM) against Bcr-Abl expressing cell lines, including most IM-resistant Bcr-Abl mutants. In a Phase I study we treated 119 pts w/IM-resistant CML in blast crisis (BC), accelerated phase (AP) or chronic phase (CP), or Ph+ ALL, Median age was 60 (range 15–83) yrs. The study design was based on the two-sample modified continual reassessment method. Initial AMN107 daily doses (mg) were: 50 qd- 7 pts, 100 qd- 7 pts, 200 qd- 10 pts, 400 qd - 10 pts, 600 qd- 6 pts, 800 qd- 19 pts, 1200 qd- 10 pts, 400 bid- 32 pts, and 600 bid- 18 pts. Intra-pts dose escalations occurred in 48/69 pts in the qd cohorts & 1/32 in the 400 bid cohort escalated to 600 bid. As of June 15, 2005, pts have been treated for 1 to 385 (median 120) days. Results for each disease group are in Table 1. AMN107 was well-tolerated. The most common drug-related adverse events were: GI: constipation G1-2 (8%), nausea G1-2 (7%), vomiting G1-2 (3%). Liver & biliary: bilirubin increased G1-2 (8%), G3 (8%), lipase elevations G 1-2 (2%), G3 (4%) G4 (1%), ALT increased G1-2 (2%), G3 (3%). Skin: pruritis G1-2 (13%), G3 (1.7%), dry skin G1-2 (12%), exanthem G1-2 (9%) G3 (1%), rash G1-2 (5%), G3 (1%). Hematologic: thrombocytopenia G3 (9%), G4 (9%), neutropenia G3 (4%), G4 (8%), anemia G1-2 (2%), G3 (4%), bone marrow aplasia (2%). General: fatigue G1-2 (4%), G3 (1%). Other notable drug-related AEs: pancreatitis G2 (2%) and CNS bleed (2%). The regimen estimated to be the MTD was 600 bid; this dose level was associated w/more neutropenia & hyperbilirubinemia. The 400 bid dose was selected for Phase II trials. Among CML pts who harbored a Bcr-Abl mutation prior to treatment, 26/43 (60%) achieved a hematologic response & 14/34 (41%) achieved a cytogenetic response. Among CML pts who had no Bcr-Abl mutation prior to Rx, 31/43 (72%) achieved a hematologic response & 17/29 (59%) achieved a cytogenetic response. Among Ph+ ALL pts who harbored a mutation prior to Rx 2/6 (33%) achieved a response & 0/2 (0%) w/no detectable mutation prior to Rx achieved a response. Pharmacokinetic (PK) studies showed steady-states reached by day 8. Cmax & AUC were dose proportional from 50 to 400 qd but plateaued w/600-1200 qd. Increased exposure was observed w/bid dosing schedules. The apparent half-life was 15 hours. AMN107 has significant activity in advanced IM-resistant CML & Ph+ ALL.Table 1DiagnosisNMedian Days of TreatmentHematologic Response*Cytogenetic Response**CML-CP161278/9 (89%)7/14 (50%)CML-Clonal Evolution91545/5 (100%)9/9 (100%)CML-AP4915434/49 (69%)14/49 (29%)CML-Myeloid BC238913/23 (57%)5/23 (22%)CML-Lymphoid BC9434/9 (44%)2/9 (22%)Ph+ALL10381/10 (10%)n/aPh+ALL minimal residual disease3561/3 (33%)n/a*pts in hematologic remission prior to enrollment were not evaluable for hematologic response **two pts were not evaluable due to partial cytogenetic response at time of enrollment

Novel Stilbene-Based Antileukemic Agents Active in P-Glycoprotein Expressing and Apoptosis-Resistant Acute Leukaemia Cell Lines.

The stilbene scaffold is a basic element for a number of biologically active natural and synthetic compounds and in accordance with Evans' definition it can be considered as a privileged structure. One of the most relevant and studied stilbenes is Resveratrol, a phytoalexin present in grapes, endowed with chemopreventive and chemotherapeutic properties and able to induce apoptosis in different cancer cell lines. Since reduced apoptosis has been implicated in the development and progression of malignant tumors and in the occurrence of chemoresistant phenotypes, resveratrol-induced apoptosis might therefore contribute to its antitumor activity. However, resveratrol is a not potent cytotoxic compound if compared with others chemotherapeutic drugs and it is scarcely active in P-glycoprotein expressing (MDR) and Bcr-Abl expressing leukaemia cells. With the aim to find new stilbene compounds active in resistant leukaemia cells we synthesized a small library of resveratrol analogs, bearing the 3,5-dimethoxy motif at the A phenyl ring and amino, methoxy and hydroxy moieties at the 3′-and/or 4′-positions. Moreover, we synthesized analogues which incorporate a phenyl ring as bioisosteric substitution of the alkenyl bridge. Among these new stilbenes we identified two compounds endowed with interesting antileukemic properties: a) a methoxylated cis derivative active at nanomolar concentrations in P-glycoprotein expressing HL60-R and CEM VBL100 acute leukaemia cell lines and in P-glycoprotein and Bcr-Abl expressing K562-ADR cell line which is resistant to apoptosis induced by most common anticancer agents, and b) a terphenyl derivative active in MDR and Bcr-Abl expressing cell lines. Both compounds induced apoptosis prevalently through the mitochondrial pathway. Differently from resveratrol and other stilbenes, the therphenyl derivative induced a block of cells in G0-G1 phase of cell cycle which was associated to the shift of the phosphorylation state of pRb from hyperphosphorylated to hypophosphorylated. Morover, low concentrations of this compound were able to induced a potent granulocytic and monocytic differentiation of HL60 cells.

Activity of AMN107, a novel aminopyrimidine inhibitor of Bcr-Abl, in imatinib-resistant bcr-abl positive lymphoid malignancies

3015 Background: AMN107, a novel aminopyrimidine ATP-competitive inhibitor of Bcr-Abl. AMN107, is 10- to 50-fold more potent than imatinib against Bcr-Abl expressing cell lines, and is effective against most cell lines expressing imatinib-resistant Bcr-Abl mutants. In two p190-Bcr-Abl ALL cell lines, AMN107 was 30–40 times more potent than imatinib in inhibiting cellular proliferation and in inhibiting phosphorylation of p190 Bcr-Abl tyrosine kinase in cell lines and primary ALL cells. Methods: In an ongoing phase I study, AMN107 was given orally, once or twice daily dosing schedule. Results: Fifteen of 67 total pts entered had lymphoid disease: lymphoid blastic phase CML (CML LBP) 7 pts; Ph+ acute lymphocytic leukemia (Ph+ ALL) 8 patients. Median age was 53 years (range 28–80). AMN doses were: QD dose cohorts (mg/day); 50 (1 pt), 100 (2 pts), 200 (3 pts), 400 (1 pt), 600 (2 pts), 800 (4 pts), 1200 (1 pt); BID dose cohort; 400 mg BID (1 pt). Pts have been treated for 15 to 89 days. In the overall cohort, possible AMN-related Grade 3 or 4 hematologic adverse events were observed in 5/67 patients (3 responded) at doses ≥ 200 mg: Possible AMN-related non-hematologic Grade 3/4 events were: 600 mg, G3 skin rash (1 pt); ≥ 800 mg, G3 SGOT and/or SGPT elevations (2 pts), G3 indirect hyperbilirubinemia (2 pts), and at 1200 mg, G2 pancreatitis (1 pt). Clinical activity has been observed in 5 of the 14 with lymphoid disease treated with doses of ≥ 600 mg: hematological improvement (1 pt, Ph+ ALL), peripheral responses (2 pts, LBP), and bone marrow responses (marrow response/no evidence of leukemia, 2 pts, LBP) 1 pt with LBP had a cytogenetic CR. Mutational data are available for 3 of the responding patients: Y253H (hematologic improvement), T315I (peripheral response), and E355G (marrow response). Pharmacokinetic (PK) studies showed steady state was generally reached by day 8. Both Cmax and AUC were dose proportional from 50 to 400 mg with a median Tmax of 3 hours. The apparent half-life was 16 hours. Conclusions: AMN107 has significant activity in patients treated with ≥ 600 mg per day in imatinib-resistant CML LBP or Ph+ ALL Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Novartis Novartis

AMN107, a novel aminopyrimidine inhibitor of Bcr-Abl, has significant activity in imatinib-resistant bcr-abl positive chronic myeloid leukemia (CML)

3014 Background: AMN107, a novel aminopyrimidine ATP-competitive inhibitor of Bcr-Abl, is 10 to 50 fold more potent than imatinib against Bcr-Abl expressing cell lines, including most imatinib-resistant Bcr-Abl mutants. The increased potency and broader activity of AMN107, relative to imatinib, may result in clinical benefit for patients (pts) with CML. Methods: In a Phase I/II study of AMN, 52 of 67 total pts (other 15 had lymphoid disease) had imatinib resistant CML in different phases. Results: Median age was 62 years (range 15–83). CML phase was: chronic (CP) 2 pts, accelerated (AP) 35 pts; myeloid blastic (BP) 15 pts. AMN daily doses were (mg/day): 50 - 6 pts, 100 - 5 pts, 200 - 7 pts, 400 - 9 pts, 600 - 1 pt, 800 - 14 pts, 1200 - 9 pts, 400 mg BID - 1 pt. Patients have been treated for 12 to 200 days. In the overall cohort, possible AMN-related Grade 3 or 4 hematologic adverse events were observed in 5/67 pts (3 responded) at doses 200 mg or more: G3 thrombocytopenia (1 pt), G4 thrombocytopenia (1 pt), G4 thrombocytopenia, G3 anemia, G4 neutropenia (1 pt), G4 neutropenia, G4 thrombocytopenia, (1 pt), and G3 neutropenia (1 pt). Possible AMN-related non-hematologic Grade 3/4 events observed: 600 mg, G3 skin rash (1 pt); ≥ 800 mg, G3 SGOT and/or SGPT elevations (2 pts), G3 indirect hyperbilirubinemia (2 pts); 1200 mg, G2 pancreatitis (1 pt). In AP, 19/30 evaluated pts (63%) responded: 11 CHR, 7 return to chronic phase (RTC) and 1 marrow CR (MR). 6/30 evaluated pts (20%) had cytogenetic (CG) responses: complete 2, partial (Ph 1–34%) 2, minor (Ph 35–90%) 2. In myeloid BP, 7/12 evaluated pts (58%) responded: 2 CHR, 5 RTC; and 1/12 (8%) had a CG response. 1 pt in CP had CHR and a CGPR. Mutational data available in 36 pts showed 9 mutations in 15 pts: G250E(4); M351T (4); E355G (2); E459Q, Y253F, F317L, F359V, T315I, M244V (1 each). 12/15 pts with mutations responded to AMN. PK studies showed steady-states reached by day 8. Both Cmax and AUC were dose proportional from 50 to 400 mg with a median Tmax of 3 hours but were not increased with 800 - 1200 mg/daily. To increase exposure, a BID schedule is being explored. The half-life was 16 hours. Conclusions: AMN107 has significant activity in imatinib-resistant CML. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Novartis

A Phase I/II Study of AMN107, a Novel Aminopyrimidine Inhibitor of Bcr-Abl, on a Continuous Daily Dosing Schedule in Adult Patients (pts) with Imatinib-Resistant Advanced Phase Chronic Myeloid Leukemia (CML) or Relapsed/Refractory Philadelphia Chromosome (Ph+) Acute Llymphocytic Leukemia (ALL).

AMN107 is a novel aminopyrimidine ATP-competitive inhibitor of Bcr-Abl. In proliferation assays AMN107 ≥10-fold more is potent than IM against Bcr-Abl expressing cell lines. AMN107 is effective against cell lines expressing the following IM-resistant Bcr-Abl mutants: Glu255Val, Phe317Leu, and Met351Thr. In addition, preliminary studies indicate that AMN107 has similar potency to IM against c-Kit and PDGFR-dependent cell proliferation. The ↑ potency and broader spectrum of activity of AMN107 against Bcr-Abl, relative to IM, may result in clinical benefit for pts with CML or Ph+ ALL. In the phase I portion of this phase I/II study, pts with IM-resistant CML-AP, CML-BC, and Ph+ ALL were eligible for treatment with AMN107 as an oral daily dose. 21 pts [median age:61 yrs (range 29–77); 15 male 6 female; performance status: 0(12 pts), 1(6 pts) or 2(3 pts)] have been enrolled in the following dose cohorts (mg/day): 50(7 pts), 100(7 pts), 200(7 pts) and have been on treatment for 8–70 days. Disease types: CML-AP (12 pts), CML-BC (6 pts), Ph+ ALL (3 pts). Intra-pts dose escalations were permitted for persistent disease in peripheral blood and/or marrow. 6/7 pts in the 50mg dose level, and 7/7 pts in the 100mg dose level have dose-escalated. No AMN107-related AE have been observed. Biologic activity, defined as at least a 50% ↓ in blasts or basophils in the peripheral blood and/or marrow lasting for at least 7 days was observed in 0/7 pts treated with 50mg/day, 4/13 pts treated with 100mg/day, and 7/15 pts treated with 200mg/day. Of the 7 pts who demonstrated biologic activity at 200mg/day, 2 pts with CML-AP had complete hematologic responses in marrow, and 1 pts with CML-AP had return to CP in marrow. Pts were not pre-selected for treatment based on mutational status of Bcr-Abl. Mutational analysis is being performed on all pts and will be correlated with response. Initial data reveal Bcr-Abl mutations in the majority (> 80%) of baseline pts samples. Preliminary data from the 50mg cohort comparing baseline peripheral blood samples with day 2 samples showed: significant ↑ in apoptosis as determined by mitochondrial potential, reduction in proliferation of CD34+ cells as measured by BrdU incorporation, and significant reductions in STAT1 phosphorylation. Reduction in CRKL phosphorylation in CD34+ cells was observed. Similar changes were noted in marrow. PK samples were collected on days 1, 2, 8, 15, 22 & 28 of cycle 1 and at time of any intra-pts dose escalation. Pharmacokinetics after 1 daily oral dose of AMN107 were characterized as little accumulation after multiple administrations with moderate inter-pts variability in exposure. Peak concentrations of AMN107 were generally achieved by 3 hrs post-dose. Preliminary PK data support 1-daily dosing. This phase I study, AMN107 given orally appears to be well tolerated with biologic effects in some pts treated with ≥100mg/day & marrow responses in some pts treated with 200mg/day. Once MTD has been determined, pts will be enrolled to multiple expansion cohorts in the phase II portion to assess activity.

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.

Chronic myelogenous leukemia blast cell proliferation is inhibited by peptides that disrupt Grb2-SoS complexes

AbstractChronic myelogenous leukemia (CML) is commonly characterized by the presence of the p210Bcr-Abl oncoprotein. Many downstream effectors of Bcr-Abl have been described, including activation of the Grb2-SoS-Ras–MAP kinase (Erk) pathway. The precise contributions of these signal-transduction proteins in CML blast cells in human patients are not yet well defined. To gain further insight into the importance of Grb2 for CML, peptides that disrupt Grb2-SoS complexes were tested. These high-affinityGrb2-binding peptides (HAGBPs) can autonomously shuttle into cells and function by binding to the N-terminal SH3 domain of Grb2. The HAGBPs were analyzed for their effects on Bcr-Abl–expressing cell lines and freshly isolated CML blast cells from patients. They induced a dramatic decrease in the proliferation of CML cell lines. This was not observed with point-mutated control peptides with abolished Grb2SH3(N) binding. As expected, Grb2-SoS complexes were greatly diminished in the HAGBP-treated cells, and MAP kinase activity was significantly reduced as determined by an activation-specific phospho-MAPK antibody. Furthermore, cell fractions that are enriched for blast cells from CML patients with active disease were also incubated with the Grb2 blocker peptides. The HAGBPs led to a significant proliferation reduction of these cells in the majority of the isolates, but not in all patients' cells. These results show that, in addition to the direct targeting of Bcr-Abl, selective inhibition of Grb2 protein complexes may be a therapeutic option for a significant number of CML patients.

Blockade of the Bcr-Abl kinase activity induces apoptosis of chronic myelogenous leukemia cells by suppressing signal transducer and activator of transcription 5-dependent expression of Bcl-xL.

Bcr-Abl-expressing leukemic cells are highly resistant to apoptosis induced by chemotherapeutic drugs. Although a number of signaling molecules have been shown to be activated by the Bcr-Abl kinase, the antiapoptotic pathway triggered by this oncogene has not been elucidated. Here, we show that the interleukin 3-independent expression of the antiapoptotic protein, Bcl-xL, is induced by Bcr-Abl through activation of signal transducer and activator of transcription (Stat)5. Inhibition of the Bcr-Abl kinase activity in Bcr-Abl-expressing cell lines and CD34(+) cells from chronic myelogenous leukemia (CML) patients induces apoptosis by suppressing the capacity of Stat5 to interact with the bcl-x promoter. Interestingly, after inhibition of the Bcr-Abl kinase, the expression of Bcl-xL is downregulated more rapidly in chronic phase than in blast crisis CML cells, suggesting an involvement of this protein in disease progression. Overall, we describe a novel antiapoptotic pathway triggered by Bcr-Abl that may contribute to the resistance of CML cells to undergo apoptosis.