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

Abstract 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.

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Constitutive Activation of SRC-Family Kinases in Chronic Myelogenous Leukemia Patients Resistant to Imatinib Mesylate in the Absence of BCR-ABL Mutations: A Rationale for Use of SRC/ABL Dual Kinase Inhibitor-Based Therapy.

Blood ◽

10.1182/blood.v106.11.1087.1087 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1087-1087 ◽

Cited By ~ 1

Author(s):

Nicholas J. Donato ◽

Ji Wu ◽

Ling-Yuan Kong ◽

Feng Meng ◽

Francis Lee ◽

...

Keyword(s):

Cell Survival ◽

Imatinib Mesylate ◽

Src Family Kinases ◽

Myelogenous Leukemia ◽

Leukemic Cells ◽

Imatinib Resistance ◽

Kinase Activation ◽

Abl Kinase ◽

Lyn Kinase ◽

Imatinib Resistant

Abstract BCR-ABL is an unregulated tyrosine kinase expressed as a consequence of a reciprocal chromosomal translocation that is common in chronic myelogenous and acute lymphocytic leukemia. BCR-ABL induces transformation of hematopoetic stem cells through tyrosine phosphorylation of multiple substrates. The src-family kinases (SFKs), Lyn and Hck, are highly activated by BCR-ABL in leukemic cells and recent studies suggest that they are substrates and essential mediators of BCR-ABL signal transduction and transformation. In cells selected for resistance to the BCR-ABL inhibitor, imatinib mesylate, Lyn kinase is overexpressed and its activation is not dependent on or regulated by BCR-ABL, suggesting that autonomous regulation of SFKs may play a role in imatinib resistant. In this report, activation of Lyn and Hck was compared in CML specimens derived from imatinib responsive and resistant patients that did not express a mutant BCR-ABL protein as their primary mediator of resistance. In imatinib sensitive cell lines and specimens derived from imatinib responsive CML patients imatinib effectively reduced activation of both BCR-ABL and SFKs. However, in multiple specimens from resistant patients, imatinib reduced BCR-ABL kinase activation but failed to reduce SFK activation. The dual ABL/SRC inhibitor, BMS-354825, blocked activation of both BCR-ABL and SFKs expressed in leukemic cells and correlated with clinical responsiveness to this agent. Animal models demonstrated that loss of imatinib-mediated inhibition of Lyn kinase activation significantly impaired its anti-tumor activity which was recovered by treatment with BMS-354825. Direct silencing of Lyn or Hck reduced CML cell survival in imatinib resistant patient specimens and cell models, suggesting a direct role for these kinases in cell survival. Our results show that SFK activation is mediated by BCR-ABL in imatinib responsive cells but these kinases escape control by BCR-ABL in CML patients that develop imatinib resistance in the absence of BCR-ABL point mutations. This form of resistance can effectively be overcome by BMS-354825 through its dual SRC and ABL kinase inhibitory activities. Dual specificity kinase inhibitors may be indicated for the treatment and prevention of imatinib resistance in CML when it is associated with constitutively activated src-family kinases.

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BCR-ABL P-Loop Deletion Detected in Imatinib-Resistant CML Patients Corresponds to Splice Variant Associated with L248V Point Mutation, Retains BCR-ABL Kinase Activity, and Responds to Dasatinib Treatment.

Blood ◽

10.1182/blood.v108.11.2168.2168 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2168-2168

Author(s):

Nikolas von Bubnoff ◽

Philipp Erben ◽

Martin Müller ◽

Tanja Lahaye ◽

Susanne Schnittger ◽

...

Keyword(s):

Splice Variant ◽

Kinase Activity ◽

Kinase Inhibitors ◽

Point Mutations ◽

Kinase Domain ◽

Imatinib Resistance ◽

Abl Kinase ◽

Imatinib Resistant ◽

Exon 4

Abstract Clonal selection of cells harboring point mutations of the BCR-ABL kinase domain are considered a major cause of resistance to imatinib. More than 40 different point mutations have been described that cause a variable degree of imatinib resistance, and display a differential response to alternative kinase inhibitors, like dasatinib or nilotinib. Here, we describe three cases (2 m, 1 f) with imatinib resistant chronic myelogenous leukemia (CML) associated with a specific deletion of 81 bp of ABL exon 4. Patients were diagnosed with chronic phase (CP) CML at the age of 52, 54, and 68 years. After initial interferon alpha based therapies for 32, 60, and 71 mo, imatinib therapy was initiated at dosages between 400–800 mg per day. After 18, 24, and 29 mo patients lost hematologic response in CP CML (n=2) or progressed to lymphoid blast crisis (BC, n=1). Molecular analysis of the ABL kinase domain revealed a deletion of a 81 bp fragment associated with a loss of amino acids 248–274 in all cases. In one patient, an additional M351T mutation was found. In the two cases with CP CML, dasatinib was commenced for imatinib resistance, resulting in a partial hematologic and minor cytogenetic response (60 and 70% Ph+ metaphases, respectively) after 14 mo of therapy. The patient with lymphoid BC was treated with vincristine and prednisone and died 24 mo after appearance of imatinib resistance. In two cases, sequencing of genomic DNA revealed an underlying CTG/GTG mutation associated with a L248V amino acid switch. The point mutation activated a cryptic splice site within ABL exon 4 leading to an in-frame splice variant characterized by the loss of a 81 bp 3′ portion of exon 4. We sought to evaluate the BCR-ABL kinase activity of the splice variant and the response to tyrosine kinase inhibitors in vitro. The 81 bp deletion of p210 BCR-ABL was cloned using cDNA from one of the patients. Using this construct, retrovirally transduced Ba/F3 cells were transformed upon growth factor withdrawal. These cells expressed BCR-ABL at the transcript and protein levels. Presence of the 81 bp deletion was confirmed by sequencing. Despite the presence of the corresponding 27 amino acid P-loop deletion (Δ248–274), Western blot indicated strong autophosphorylation of BCR-ABL, which decreased in the presence of imatinib to non-detectable levels at concentrations of 1.25μM and above. In the presence of imatinib/nilotinib/dasatinib, the growth of BCR-ABL expressing Ba/F3 cells was shifted from an IC50 of 125/30/0.5nM for wild-type BCR-ABL to 470/185/1.9nM for Δ248–274 cells. Thus, in vitro data demonstrate that deletion of almost the entire P-loop does not abrogate BCR-ABL kinase activity and results in only marginal resistance towards ABL kinase inhibitors. We conclude that deletions of BCR-ABL may be the result of alternative splicing generated by point mutations associated with resistance to imatinib. The Δ248–274 splice variant retains BCR-ABL kinase activity and sensitivity to imatinib, nilotinib, and dasatinib.

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Lyn Kinase Alters Gab2 Phosphorylation and c-Cbl Protein Levels To Regulate Imatinib Sensitivity and Survival of Chronic Myelogenous Leukemia Cells.

Blood ◽

10.1182/blood.v108.11.2132.2132 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2132-2132

Author(s):

Ji Wu ◽

Feng Meng ◽

Moshe Talpaz ◽

Nicholas J. Donato

Keyword(s):

Tyrosine Phosphorylation ◽

Kinase Inhibitor ◽

K562 Cells ◽

Imatinib Resistance ◽

Kinase Inhibition ◽

Protein Levels ◽

Abl Kinase ◽

Lyn Kinase ◽

Imatinib Resistant ◽

Cbl Protein

Abstract The tyrosine kinase inhibitor imatinib mesylate (Gleevec) is effective in controlling BCR-ABL expressing leukemias but resistance occurs in some early phase patients while it is more common in advanced disease. Resistance has been generally associated with mutations in the BCR-ABL kinase that effect drug affinity. However patients are also increasingly reported to fail imatinib therapy while retaining wild-type BCR-ABL expression. Our previous studies suggested a role for Lyn, a Src-related kinase, in imatinib resistance. K562 cells selected for imatinib resistance (K562R) overexpress Lyn kinase and its targeted silencing overcomes imatinib resistance and engages apoptosis. Overexpression of Lyn in K562 cells reduces imatinib sensitivity (3-fold) and patients that fail imatinib therapy in the absence of BCR-ABL mutations express a highly activated Lyn kinase that is not suppressed by imatinib. Silencing Lyn expression in patient specimens induces changes in cell survival that are proportional to the level of Lyn protein reduction. To understand the role of Lyn kinase in imatinib resistance and apoptosis we examined proteins associated with this kinase in imatinib resistant cell lines, leukemic cells overexpressing Lyn and specimens derived from imatinib resistant patients. Lyn overexpression blocked complete suppression of BCR-ABL tyrosine phosphorylation by imatinib and affected BCR-ABL signaling adaptors. Although BCR-ABL forms a stable complex with the leukemogenic-critical adaptor protein Gab2 in imatinib sensitive cells, Lyn overexpression resulted in the formation of Lyn:Gab2 complexed in resistant cells. BCR-ABL kinase inhibition failed to reduce tyrosine phosphorylation of Gab2 in these cells while Lyn silencing or kinase inhibition (with dasatinib) completely suppressed Gab2 tyrosine phosphorylation and correlated with the induction of apoptosis. Lyn silencing in K562R cells also lead to a reciprocal increase in the tyrosine phosphorylation and association with a protein of ~120kDa, identified as the E3 ligase, c-Cbl. Lyn overexpression in K562 cells reduced their imatinib sensitivity and reduced c-Cbl protein levels. Kinase inhibitor and co-transfection studies demonstrated that tyrosine phosphorylation of c-Cbl at a critical signaling site (Y774) is primarily controlled by BCR-ABL and deletion or mutation of the c-Cbl RING domain altered its BCR-ABL phosphorylation. These results suggest that c-Cbl complexes are regulated at both the protein and phosphorylation level by Lyn and BCR-ABL kinase activities, respectively. Overexpression and/or activation of Lyn may disrupt the balance and regulation of critical regulators of leukemogenic signaling (Gab2) or protein trafficking and stability (c-Cbl), resulting in increased cell survival and reduced responsiveness to BCR-ABL kinase inhibition. We conclude that Lyn alters the level and function of critical signaling adaptor proteins in CML cells.

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Correlation of Clinical Response to Nilotinib with BCR-ABL Mutation Status in Advanced Phase Chronic Myelogenous Leukemia (CML-AP) Patients with Imatinib-Resistance or Intolerance.

Blood ◽

10.1182/blood.v110.11.1940.1940 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1940-1940 ◽

Cited By ~ 2

Author(s):

Giuseppe Saglio ◽

Dong-Wook Kim ◽

Andreas Hochhaus ◽

Simona Soverini ◽

P. Erben ◽

...

Keyword(s):

Disease Progression ◽

Cytogenetic Response ◽

Kinase Domain ◽

Mutant Clone ◽

Imatinib Resistance ◽

Mutation Status ◽

Abl Kinase ◽

Imatinib Resistant ◽

Kinase Domain Mutations

Abstract The 2nd-generation bcr-abl inhibitor nilotinib is more potent than imatinib (IC50 <30 nM) against unmutated bcr-abl and active against 32/33 imatinib-resistant BCR-ABL mutants in vitro. We investigated the in vivo activity of nilotinib stratified by the baseline BCR-ABL mutation status in 127 imatinib-resistant or -intolerant CML-AP patients (pts) enrolled in an open-label phase II trial of nilotinib. Eighty-five pts (85/127, 67%) were screened prior to nilotinib therapy for BCR-ABL kinase domain mutations by direct sequencing. Of the 85 pts, 75 (88%) were resistant to imatinib and 10 (12%) were intolerant using standard published criteria. Twenty-two different baseline mutations involving 19 amino acids were identified in 50 (59%) pts analyzed. Other 35 (41%) pts did not have a baseline mutation. The most frequent mutation types identified included M351T (8 pts), G250E (7 pts), Y253H (6 pts), M244V (5 pts), F359V (5 pts) and T315I (5 pts). Twenty-two percent of pts with baseline mutations (11/50) showed more than one mutation (9 with two, 1 with three, and 1 with four mutations). All baseline mutations occurred in imatinib-resistant pts but none in intolerant pts. After 12 months of therapy, confirmed (confirmed in two consecutive analyses 4 week apart) hematologic response (HR) was achieved in 48% (21/50), major cytogenetic response (MCR) in 20% (10/50), and complete cytogenetic response (CCR) in 16% (8/50) of imatinib-resistant pts with baseline mutation versus 44% (12/25), 40% (10/25), and 20% (2/25) of imatinib-resistant pts without baseline mutation, respectively. Responses appeared to be affected by the in vitro sensitivity of the mutant clone against nilotinib. Pts with less sensitive mutation (cellular IC50 of >200nM: Y253H, E255K, E255V, F359C) representing 13% (11/85) of all patients assessed for baseline mutation, showed 13% (1/11) HR and 13% (1/11) MCyR compared to 74% (17/28) and 18% (5/28) respectively in the mutant group with IC50 of ≤200 nM. The nilotinib resistant T315I mutation occurred in 5 pts. Only one of these 5 pts who had T315I and G250E dual mutation achieved HR conceivably reflecting the sensitivity of G250E or non-mutant clone to nilotinib. At the time of data analyses, 50% of pts with baseline mutation were free of disease progression versus 62% of pts without baseline mutation. Rate of progression was 64% (7/11) in the group with less sensitive mutations and 60% (3/5) in pts. with T315I. However, the mutants most frequently associated with progression were F359V and M244V both having 4/5 pts (80%) progressed. In summary, BCR-ABL kinase domain mutations were identified at baseline in 59% of all pts in this cohort and in 67% of pts with imatinib resistance. Responses were observed across a broad spectrum of mutant genotypes. The rate of responses and disease progression may be affected by the baseline mutation types, although a larger data set with longer follow up is needed to further establish the correlation.

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To Study Resistant Mechanisms and Reversal In An Imatinib Resistant Ph+ Positive Acute Lymphoblastic Leukemia Cell Line

Blood ◽

10.1182/blood.v116.21.2135.2135 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2135-2135

Author(s):

Hongyun Xing ◽

Yuping Gong ◽

Ting Liu

Keyword(s):

Cell Signaling ◽

Cell Line ◽

Lymphoblastic Leukemia ◽

Kinase Domain ◽

Rt Pcr ◽

Imatinib Resistance ◽

Abl Kinase ◽

Imatinib Resistant ◽

Cell Signaling Pathways

Abstract Abstract 2135 Objective To establish an imatinib resistant Bcr-Abl positive acute lymphoblastic leukemia (ALL) cell line in vitro and to study imatibin resistance in Ph+ ALL. The reversal of the imatinib resistance by rapamycin, the second generation tyrosine kinase inhibitor and proteasome inhibitor was studied. Methods Ph(+) ALL SUP-B15 cell line was cultured in gradually increasing concentrations of imatinib to generate the imatinib resistant cell line at 6 μM imatinib. The cytotoxic effect of imatinib and other drugs was analyzed by MTT assay. RT-PCR, flow cytometry, Western blot analyses of proteins, DNA sequence analysis of ABL kinase domain were used to clarify the possible mechanisms of the imatinib resistance in the SUP-B15/RI cell line. Results We established the imatinib resistant Ph+ ALL cell line. The fusion bcr-abl gene was 6.1 times as high as that of the parental sensitive cell, and the mdr1 gene also increased 1.7 times in SUP-B15/RI cell line by the RT-PCR detection. However, the expression of hoct1 Abcl–2 and topoIIα gene were no difference between two cell lines by the RT-PCR detection. A K362S point mutation in the Abl kinase domain of SUP-B15/RI was found. The detection of cell signaling pathway of PI3K/AKT/mTOR, RAS/RAF, NF-κBA JNK and STAT showed the expression of PTEN and 4EBP-1 was down-regulated, AKT, mTOR and P70S6K was up-regulated and the expression of other cell signaling pathways in SUP-B15/RI was similar to its parental sensitive cell line. Dasatinib, nilotinib, and bortezomib could inhibit proliferation of SUP-B15/RI cells at nM concentration. SUP-B15/RI cell line also showed partial resistance to dasatinib and nilotinib, but not bortezomib. The combination of imatinib with rapamycin had synergistic effect to the resistance cell line. Conclusion In vitro, we establish imatinib resistant Ph + ALL cell line. Overexpression of bcr-abl and mdr1 gene, K362S point mutation in ABL kinase domain and up-regulation of the cell signaling pathways of PI3K/AKT/mTOR, RAS/RAF in SUP-B15/RI cell line were involved in the resistance mechanisms. The SUP-B15/RI cell line was also resistant to the second generation tyrosine kinaeses dasatinib and nilotinib,not bortezomib in vitro. However, the combination of imatinib with rapamycin can partially overcome the resistance. Blockade of the ubiquitin-proteasome could be a promising pathway to overcome resistance to imatinib. Disclosures: No relevant conflicts of interest to declare.

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p27 Is Mislocalized to the Cytoplasm by BCR-ABL In a Kinase-Independent Manner and Contributes to Leukemogenesis

Blood ◽

10.1182/blood.v116.21.512.512 ◽

2010 ◽

Vol 116 (21) ◽

pp. 512-512

Author(s):

Anupriya Agarwal ◽

Ryan J Meckenzie ◽

Thomas O'Hare ◽

Kavin B Vasudevan ◽

Dorian H LaTocha ◽

...

Keyword(s):

Tumor Suppressor ◽

Kinase Activity ◽

Immunofluorescence Microscopy ◽

Independent Effect ◽

Histopathological Analysis ◽

Imatinib Treatment ◽

Wild Type ◽

Abl Kinase

Abstract Abstract 512 Background: BCR-ABL promotes cell cycle progression by interfering with the regulatory functions of p27, a cyclin dependent kinase (Cdk) inhibitor and tumor suppressor. We have previously shown that BCR-ABL kinase activity promotes degradation of nuclear p27 (Agarwal, A. et al. Blood 2008). Additionally, in primary CML cells, p27 is mislocalized to the cytoplasm, thereby relieving Cdks from p27 inhibition. Results from studies of solid tumors show that cytoplasmic p27 can actively contribute to oncogenesis, raising the question of whether cytoplasmic p27 in CML cells may actively promote leukemogenesis rather than merely compromise Cdk inhibition. We hypothesize that BCR-ABL disrupts p27 function in a dual manner by reducing nuclear p27, where p27 normally serves as a tumor suppressor, and by increasing cytoplasmic p27, where it might have oncogenic activity. Experimental Approach and Results: Immunoblotting of nuclear and cytoplasmic lysates of CD34+ cells from 11 CML patients revealed that p27 localization is predominantly cytoplasmic in the majority of patients (10/11; 91%) irrespective of disease phase, while p27 was mostly nuclear in normal controls. Similar results were obtained by immunofluorescence microscopy. Imatinib treatment increased nuclear p27 suggesting that nuclear p27 levels are regulated by BCR-ABL kinase activity. However, imatinib does not alter cytoplasmic p27 levels, suggesting that cytoplasmic mislocalization of p27 is a kinase-independent effect of BCR-ABL. Kinase-independent regulation of cytoplasmic p27 localization was also tested by immunofluorescence microscopy of p27−/− MEFs engineered to express active or kinase-dead BCR-ABL in combination with wild-type p27. In these cells cytoplasmic p27 abundance was increased both by kinase-active or kinase-dead BCR-ABL as compared to the vector control. To interrogate the role of p27 in vivo we retrovirally transduced p27+/+ or p27−/− bone marrow with BCR-ABL-GFP retrovirus and sorted Lin-/c-Kit+/Sca-I+ cells by FACS, allowing for injection of exactly matched numbers of BCR-ABL-expressing GFP+ cells (5000/animal). Median survival was significantly reduced for recipients of p27−/− marrow as compared to p27+/+ controls (34 days vs. 93 days p<0.0001). Recipients of p27−/− marrow also exhibited significantly increased white blood cell (4.5-fold) and platelet counts (3.9-fold) as well as spleen size (6-fold) and liver size (1.6-fold). Accordingly, there was more pronounced leukemic infiltration of myeloid precursors on histopathology as compared to controls. An in vivo competition experiment performed by injecting equal numbers of BCR-ABL-transduced p27−/− and p27+/+ marrow cells in congenic recipients resulted in leukemias in recipient mice (N=8) that were derived exclusively from p27−/− cells. In total, these results suggest that the net function of p27 in CML is tumor suppressive. To functionally dissect the role of nuclear and cytoplasmic p27, we used p27T187A transgenic mice (in which nuclear p27 degradation is reduced) and p27S10A mice (in which p27 export to the cytoplasm is reduced resulting in predominantly nuclear p27). Mice of matched genetic background were used as p27WT controls in CML retroviral transduction/transplantation experiments. In both cases, survival was prolonged compared to controls: 25 vs. 21 days for p27T187A (p=0.05) and 32 vs. 23 days for p27S10A (p=0.01). This suggests that stabilization of nuclear p27 (p27T187A) and more significantly lack of cytoplasmic p27 (p27S10A) attenuate BCR-ABL-mediated leukemogenesis. Consistent with this, autopsy and histopathological analysis revealed reduced hepatosplenomegaly (p27T187A mice) and improved cell differentiation with a relative increase of mature neutophils (p27S10A mice) as compared to wild-type controls. Conclusions: These results provide in vivo evidence that p27 has genetically separable dual roles in CML as both a nuclear tumor suppressor and cytoplasmic oncogene. A kinase-independent activity of BCR-ABL contributes to leukemogenesis through aberrant p27 localization to the cytoplasm. This oncogene activity is independent from the kinase-dependent degradation of nuclear p27. We speculate that the inability of tyrosine kinase inhibitors to reverse cytoplasmic p27 mislocalization may contribute to disease persistence despite effective inhibition of BCR-ABL kinase activity. Disclosures: Deininger: Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; genzyme: Research Funding.

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Dual Inhibition of Bcr-Abl and Hsp90 By C086 Potently Inhibits the Proliferation of Imatinib-Resistant CML Cells

Blood ◽

10.1182/blood.v124.21.914.914 ◽

2014 ◽

Vol 124 (21) ◽

pp. 914-914

Author(s):

Lixian Wu ◽

Jing Yu ◽

Yang Liu ◽

Lou Liguang ◽

Yong Wu ◽

...

Keyword(s):

Kinase Activity ◽

Conflicts Of Interest ◽

Chaperone Function ◽

Abl Kinase ◽

Fluorescence Measurements ◽

Biochemical Assays ◽

Imatinib Resistant ◽

Hsp90 Chaperone ◽

Hsp90 Function

Abstract Purpose: Although such tyrosine kinase inhibitors (TKIs) as imatinib provide an effective treatment against Bcr-Abl kinase activity in the mature cells of CML patients, TKIs probably cannot eradicate the leukemia stem cell (LSC) population. Therefore, alternative therapies are required to target both mature CML cells with wild-type (WT) or mutant Bcr-Abl and LSCs. To investigate the effect of C086, a derivative of curcumin, on imatinib-resistant cells, we explored its underlying mechanisms of Bcr-Abl kinase and heat shock protein 90 (Hsp90) function inhibition. Experimental Design: Biochemical assays were used to test ABL kinase activity; fluorescence measurements using recombinant NHsp90, Hsp90 ATPase assay, immunoprecipitation and immunoblotting were applied to examine Hsp90 function; Colony-forming unit (CFU), long-term culture-initiating cells (LTC-ICs), and flow cytometry were used to test CML progenitor and stem cells. Results: Biochemical assays with purified recombinant Abl kinase confirmed that C086 can directly inhibit the kinase activity of Abl, including WT and the Q252H, Y253F, and T315I mutations. Furthermore, we identified C086 as a novel Hsp90 inhibitor with the capacity to disrupt the Hsp90 chaperone function in CML cells. Consequently, inhibited the growth of both imatinib-sensitive and resistant CML cells. Interestingly, C086 has the capacity to inhibit LTC-ICs and to induce apoptosis in both CD34+CD38+ and CD34+CD38- cells in vitro. Moreover, C086 could decreased the number of CD45+, CD45+CD34+CD38+ and CD45+CD34+CD38- cells in CML NOD-SCID mice. Conclusions: Dual suppression of Abl kinase activity and Hsp90 chaperone function by C086 provides a new therapeutic strategy for treating Bcr-Abl-induced leukemia resistant to TKIs. Disclosures No relevant conflicts of interest to declare.

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BCR-ABL truncation due to premature translation termination as a mechanism of resistance to kinase inhibitors

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.7028 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. 7028-7028

Author(s):

C. Yeh ◽

W. Ma ◽

H. Kantarjian ◽

Z. J. Zhang ◽

J. Cortes ◽

...

Keyword(s):

Cell Line ◽

Peripheral Blood ◽

Myeloid Leukemia ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Kinase Domain ◽

Leukemic Cells ◽

Leukemia Cell Line ◽

Imatinib Resistance ◽

Abl Kinase

7028 Background: The major mechanism underlying imatinib resistance in patients with chronic myeloid leukemia (CML) is clonal expansion of leukemic cells with point mutations in the BCR-ABL tyrosine kinase. We describe three novel ABL premature termination mutations leading to BCR-ABL truncation in leukemia patients with multidrug (imatinib/nilotinib/dasatinib) resistance. Methods: Peripheral blood or bone marrow samples from drug-resistant CML patients were collected. Total nucleic acids were purified and subjected to two rounds of PCR analysis, with the first PCR designed to eliminate amplification of the wild-type, non-translocated ABL gene. Bi-directional sequencing was then performed. HL60 cells (a Ph-negative myeloid leukemia cell line) and peripheral blood of healthy subjects were used as negative controls; a human CML cell line (K562) was used as a positive control. Results: We identified an exon 7 deletion in three CML patients, a 4-nt insertion (908insCAGG) near the exon 5/6 junction in one CML case, and an exon 6 point mutation (997C>T) in one patient with acute lymphoblastic leukemia (ALL). These mutations all create premature stop codons and cause termination at residues 381, 315, and 333, respectively, leading to truncated proteins with only the first quarter of the kinase domain (P-loop) or lacking the C-terminus of ABL including the A-loop. Conclusions: These novel mutations, and the previously documented 35-nt insertion in exon 8, may constitute a new class of mutations that 1) cause truncation of the BCR-ABL kinase; (2) abolish the regulatory element in the ABL kinase domain and the downstream C-terminal region; and (3) confer significant drug resistance. [Table: see text]

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Inhibition of the ABL Kinase Activity Blocks the Proliferation of BCR/ABL+Leukemic Cells and Induces Apoptosis

Blood Cells Molecules and Diseases ◽

10.1006/bcmd.1997.0155 ◽

1997 ◽

Vol 23 (3) ◽

pp. 380-394 ◽

Cited By ~ 211

Author(s):

Carlo Gambacorti-Passerini ◽

Philipp le Coutre ◽

Luca Mologni ◽

Mirco Fanelli ◽

Carla Bertazzoli ◽

...

Keyword(s):

Kinase Activity ◽

Leukemic Cells ◽

Abl Kinase

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A Novel Mechanism for Imatinib Mesylate (STI571) Resistance in CML Cells: Contribution of TC-PTP to Modulating Signals Down-Stream from the BCR-ABL Fusion Protein.

Blood ◽

10.1182/blood.v104.11.1984.1984 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1984-1984

Author(s):

Takatsune Shimizu ◽

Yoshitaka Miyakawa ◽

Satoshi Iwata ◽

Akiko Kuribara ◽

Tony Tiganis ◽

...

Keyword(s):

Protein Kinase ◽

Imatinib Mesylate ◽

Acquired Resistance ◽

Myelogenous Leukemia ◽

Cell Protein ◽

Imatinib Treatment ◽

Apoptotic Cells ◽

Imatinib Resistance ◽

Imatinib Resistant ◽

Proliferation Assays

Abstract Acquired resistance to imatinib mesylate (STI571) in chronic myelogenous leukemia (CML) patients has become a serious problem. To adress the novel molecular mechanism for imatinib-resistance in CML, we previously established imatinib-resistant sublines (designated KTR cells) from the CML cell line KT-1. We have analyzed p-glycoprotein expression, the number of bcr-abl fusion gene and the sequence of ATP binding site of ABL kinase domain. However, these were not responsible for imatinib-resistance in KTR cells. Interestingly, T-cell protein tyrosine phosphatase (TC-PTP) protein levels were markedly down-regulated in all KTR cells as compared to parental KT-1 cells. Therefore, we examined whether the suppression of TC-PTP expression might contribute to imatinib-resistance in KTR cells. We transduced the nuclear isoform of TC-PTP (TC45) and catalytically inactive TC45-D182A cDNA into KTR cells by retroviral gene transfer. Subsequently, we analyzed the sensitivity to imatinib by MTT proliferation assays. We also studied the signaling pathways in all transduced cells by Western blottings. KTR cells successfully expressed TC45 and TC45-D182A protein (designated KTR-TC45 and KTR-D182A cells, respectively). In MTT proliferation assays, the proliferation of KTR-TC45 cells restored their sensitivity to imatinib, but not in KTR-mock or KTR-D182A cells, indicating that transduced catalytically active TC45 restored the sensitivity to imatinib in KTR cells. In KTR2-mock cells, the percentage of annexin V positive apoptotic cells was 8% in the control and was increased to 25% upon imatinib treatment. In KTR-TC45 cells, the percentage of apoptotic cells was increased from 12% to 56% by the treatment with imatinib, suggesting that TC45 expression in KTR cells restored the susceptibility to apoptosis by imatinib mesylate. Taken together, these results indicate that the sensitivity to imatinib in KTR cells can be modulated by TC-PTP expression. In parental KT-1 cells, phosphorylation of STAT5 was abolished with the treatment of 0.5 μM imatinib for 1 hour. In contrast, STAT5 phosphorylation in KTR cells was stronger than that of KT-1 cells and only slightly suppressed upon exposure to 0.5 μM imatinib. In KTR-mock and KTR-D182A transduced cells, STAT5 phosphorylation was augmented compared to KTR-TC45 transduced cells. Upon treatment with 0.5 μM imatinib for 1 hour, phosphorylation of STAT5 was abolished in KTR2-TC45 cells whereas it remained elevated in KTR-mock and KTR-D182A cells. The expression of TC-PTP had no effect on the phosphorylation of the JAK2 or BCR-ABL in KTR cells. Besides, expression of TC-PTP did not alter protein kinase PKB/AKT or mitogen-activated protein kinase signaling in KTR cells. These results indicate that the loss of TC-PTP could enhance tyrosine phosphorylation of STAT5 and was involved in the acquired resistance to imatinib in KTR cells. In conclusion, we demonstrated that reconstitution of TC-PTP in imatinib-resistant KTR cells restored the sensitivity to imatinib. Although it will be necessary to ascertain the relevance of our studies in primary samples, we would like to propose that the loss of TC-PTP may represent a novel mechanism by which CML cells can acquire imatinib-resistance.

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