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1.  The ABL switch control inhibitor DCC-2036 is active against the chronic myeloid leukemia mutant BCR-ABLT315I and exhibits a narrow resistance profile 
Cancer research  2011;71(9):3189-3195.
Acquired point mutations within the BCR-ABL kinase domain represent a common mechanism of resistance to ABL inhibitor therapy in patients with chronic myeloid leukemia (CML). The BCR-ABLT315I mutant is highly resistant to imatinib, nilotinib, and dasatinib and is frequently detected in relapsed patients. This critical gap in resistance coverage drove development of DCC-2036, an ABL inhibitor which binds the switch control pocket involved in conformational regulation of the kinase domain. We evaluated the efficacy of DCC-2036 against BCR-ABLT315I and other mutants in cellular and biochemical assays and conducted cell-based mutagenesis screens. DCC-2036 inhibited autophosphorylation of ABL and ABLT315I enzymes, and this activity was consistent with selective efficacy against Ba/F3 cells expressing BCR-ABL (IC50: 19 nmol/L), BCR-ABLT315I (IC50: 63 nmol/L), and most kinase domain mutants. Ex vivo exposure of CML cells from patients harboring BCR-ABL or BCR-ABLT315I to DCC-2036 revealed marked inhibition of colony formation and reduced phosphorylation of the direct BCR-ABL target CrkL. Cell-based mutagenesis screens identified a resistance profile for DCC-2036 centered around select P-loop mutations (G250E, Q252H, Y253H, E255K/V), although a concentration of 750 nmol/L DCC-2036 suppressed the emergence of all resistant clones. A decreased concentration of DCC-2036 (160 nmol/L) in dual-combination with either nilotinib or dasatinib achieved the same zero outgrowth result. Further screens for resistance due to BCR-ABL compound mutations (two mutations in the same clone) identified BCR-ABLE255V / T315I as the most resistant mutant. Taken together, these findings support continued evaluation of DCC-2036 as an important new agent for treatment-refractory CML.
doi:10.1158/0008-5472.CAN-10-3224
PMCID: PMC3206627  PMID: 21505103
BCR-ABL; imatinib resistance; DCC-2036
2.  Destabilization of Bcr-Abl/Jak2 Network by a Jak2/Abl Kinase Inhibitor ON044580 Overcomes Drug Resistance in Blast Crisis Chronic Myelogenous Leukemia (CML) 
Genes & cancer  2010;1(4):346-359.
Bcr-Abl is the predominant therapeutic target in chronic myeloid leukemia (CML), and tyrosine kinase inhibitors (TKIs) that inhibit Bcr-Abl have been successful in treating CML. With progression of CML disease especially in blast crisis stage, cells from CML patients become resistant to imatinib mesylate (IM) and other TKIs, resulting in relapse. Because Bcr-Abl is known to drive multiple signaling pathways, the study of the regulation of stability of Bcr-Abl in IM-resistant CML cells is a critical issue as a possible therapeutic strategy. Here, we report that a new dual-kinase chemical inhibitor, ON044580, induced apoptosis of Bcr-Abl+ IM-sensitive, IM-resistant cells, including the gatekeeper Bcr-Abl mutant, T315I, and also cells from blast crisis patients. In addition, IM-resistant K562-R cells, cells from blast crisis CML patients, and all IM-resistant cell lines tested had reduced ability to form colonies in soft agar in the presence of 0.5 µM ON044580. In in vitro kinase assays, ON044580 inhibited the recombinant Jak2 and Abl kinase activities when the respective Jak2 and Abl peptides were used as substrates. Incubation of the Bcr-Abl+ cells with ON044580 rapidly reduced the levels of the Bcr-Abl protein and also reduced the expression of HSP90 and its client protein levels. Lysates of Bcr-Abl+ cell lines were found to contain a large signaling network complex composed of Bcr-Abl, Jak2, HSP90, and its client proteins as detected by a gel filtration column chromatography, which was rapidly disrupted by ON044580. Therefore, targeting Jak2 and Bcr-Abl kinases is an effective way to destabilize Bcr-Abl and its network complex, which leads to the onset of apoptosis in IM-sensitive and IM-resistant Bcr-Abl+ cells. This inhibitory strategy has potential to manage all types of drug-resistant CML cells, especially at the terminal blast crisis stage of CML, where TKIs are not clinically useful.
doi:10.1177/1947601910372232
PMCID: PMC2927857  PMID: 20798787
CML; Bcr-Abl; Jak2; drug resistance; apoptosis
3.  Destabilization of Bcr-Abl/Jak2 Network by a Jak2/Abl Kinase Inhibitor ON044580 Overcomes Drug Resistance in Blast Crisis Chronic Myelogenous Leukemia (CML) 
Genes & Cancer  2010;1(4):346-359.
Bcr-Abl is the predominant therapeutic target in chronic myeloid leukemia (CML), and tyrosine kinase inhibitors (TKIs) that inhibit Bcr-Abl have been successful in treating CML. With progression of CML disease especially in blast crisis stage, cells from CML patients become resistant to imatinib mesylate (IM) and other TKIs, resulting in relapse. Because Bcr-Abl is known to drive multiple signaling pathways, the study of the regulation of stability of Bcr-Abl in IM-resistant CML cells is a critical issue as a possible therapeutic strategy. Here, we report that a new dual-kinase chemical inhibitor, ON044580, induced apoptosis of Bcr-Abl+ IM-sensitive, IM-resistant cells, including the gatekeeper Bcr-Abl mutant, T315I, and also cells from blast crisis patients. In addition, IM-resistant K562-R cells, cells from blast crisis CML patients, and all IM-resistant cell lines tested had reduced ability to form colonies in soft agar in the presence of 0.5 µM ON044580. In in vitro kinase assays, ON044580 inhibited the recombinant Jak2 and Abl kinase activities when the respective Jak2 and Abl peptides were used as substrates. Incubation of the Bcr-Abl+ cells with ON044580 rapidly reduced the levels of the Bcr-Abl protein and also reduced the expression of HSP90 and its client protein levels. Lysates of Bcr-Abl+ cell lines were found to contain a large signaling network complex composed of Bcr-Abl, Jak2, HSP90, and its client proteins as detected by a gel filtration column chromatography, which was rapidly disrupted by ON044580. Therefore, targeting Jak2 and Bcr-Abl kinases is an effective way to destabilize Bcr-Abl and its network complex, which leads to the onset of apoptosis in IM-sensitive and IM-resistant Bcr-Abl+ cells. This inhibitory strategy has potential to manage all types of drug-resistant CML cells, especially at the terminal blast crisis stage of CML, where TKIs are not clinically useful.
doi:10.1177/1947601910372232
PMCID: PMC2927857  PMID: 20798787
CML; Bcr-Abl; Jak2; drug resistance; apoptosis
4.  Overcoming Bcr-Abl T315I mutation by combination of GNF-2 and ATP competitors in an Abl-independent mechanism 
BMC Cancer  2012;12:563.
Background
Philadelphia positive leukemias are characterized by the presence of Bcr-Abl fusion protein which exhibits an abnormal kinase activity. Selective Abl kinase inhibitors have been successfully established for the treatment of Ph (+) leukemias. Despite high rates of clinical response, Ph (+) patients can develop resistance against these kinase inhibitors mainly due to point mutations within the Abl protein. Of special interest is the ‘gatekeeper’ T315I mutation, which confers complete resistance to Abl kinase inhibitors. Recently, GNF-2, Abl allosteric kinase inhibitor, was demonstrated to possess cellular activity against Bcr-Abl transformed cells. Similarly to Abl kinase inhibitors (AKIs), GNF-2 failed to inhibit activity of mutated Bcr-Abl carrying the T315I mutation.
Methods
Ba/F3 cells harboring native or T315I mutated Bcr-Abl constructs were treated with GNF-2 and AKIs. We monitored the effect of GNF-2 with AKIs on the proliferation and clonigenicity of the different Ba/F3 cells. In addition, we monitored the auto-phosphorylation activity of Bcr-Abl and JAK2 in cells treated with GNF-2 and AKIs.
Results
In this study, we report a cooperation between AKIs and GNF-2 in inhibiting proliferation and clonigenicity of Ba/F3 cells carrying T315I mutated Bcr-Abl. Interestingly, cooperation was most evident between Dasatinib and GNF-2. Furthermore, we showed that GNF-2 was moderately active in inhibiting the activity of JAK2 kinase, and presence of AKIs augmented GNF-2 activity.
Conclusions
Our data illustrated the ability of allosteric inhibitors such as GNF-2 to cooperate with AKIs to overcome T315I mutation by Bcr-Abl-independent mechanisms, providing a possibility of enhancing AKIs efficacy and overcoming resistance in Ph+ leukemia cells.
doi:10.1186/1471-2407-12-563
PMCID: PMC3561207  PMID: 23186157
Philadelphia chromosome; Bcr-Abl; “gatekeeper” mutation T315I; Allosteric inhibition; Abl kinase inhibitors
5.  AMN107 (nilotinib): a novel and selective inhibitor of BCR-ABL 
British Journal of Cancer  2006;94(12):1765-1769.
Chronic myelogenous leukaemia (CML) and Philadelphia chromosome positive (Ph+) acute lymphoblastic leukaemia (ALL) are caused by the BCR-ABL oncogene. Imatinib inhibits the tyrosine kinase activity of the BCR-ABL protein and is an effective, frontline therapy for chronic-phase CML. However, accelerated or blast-crisis phase CML patients and Ph+ ALL patients often relapse due to drug resistance resulting from the emergence of imatinib-resistant point mutations within the BCR-ABL tyrosine kinase domain. This has stimulated the development of new kinase inhibitors that are able to over-ride resistance to imatinib. The novel, selective BCR-ABL inhibitor, AMN107, was designed to fit into the ATP-binding site of the BCR-ABL protein with higher affinity than imatinib. In addition to being more potent than imatinib (IC50<30 nM) against wild-type BCR-ABL, AMN107 is also significantly active against 32/33 imatinib-resistant BCR-ABL mutants. In preclinical studies, AMN107 demonstrated activity in vitro and in vivo against wild-type and imatinib-resistant BCR-ABL-expressing cells. In phase I/II clinical trials, AMN107 has produced haematological and cytogenetic responses in CML patients, who either did not initially respond to imatinib or developed imatinib resistance. Dasatinib (BMS-354825), which inhibits Abl and Src family kinases, is another promising new clinical candidate for CML that has shown good efficacy in CML patients. In this review, the early characterisation and development of AMN107 is discussed, as is the current status of AMN107 in clinical trials for imatinib-resistant CML and Ph+ ALL. Future trends investigating prediction of mechanisms of resistance to AMN107, and how and where AMN107 is expected to fit into the overall picture for treatment of early-phase CML and imatinib-refractory and late-stage disease are discussed.
doi:10.1038/sj.bjc.6603170
PMCID: PMC2361347  PMID: 16721371
BCR-ABL; AMN107; nilotinib; dasatinib; imatinib-resistance
6.  Nilotinib based pharmacophore models for BCRABL 
Bioinformation  2012;8(14):658-663.
Tyrosine kinase inhibitors have revolutionized the treatment of several malignancies, converting lethal diseases in a manageable aspect. Imitanib, a small molecule ABL kinase inhibitor is a highly effective therapy for early phase chronic myeloid leukemia (CML), which has constitutively active ABL kinase activity owing to the over expression of the BCR-ABL fusion protein. But some patients develop imatinib resistance, particularly in the advanced phases of CML.The discovery of resistance mechanisms of imitanib; urge forward the development of second generation drugs. Nilotinib, a second generation drug is more potent inhibitor of BCR-ABL than imatinib. But nilotinib also develops dermatologic events and headache in patients. Large information about BCR-ABL structure and its inhibitors are now available. Based on the pharmacophore modeling approaches, it is possible to decipher the molecular determinants to inhibit BCR-ABL. We conducted a structure based and ligand based study to identify potent natural compounds as BCR-ABL inhibitor. First kinase inhibitors were docked with the receptor (BCR-ABL) and nilotinib was selected as a pharmacophore due its high binding efficiency. Eleven compounds were selected out of 1457 substances which have mutual pharmacopohre features with nilotinib. These eleven compounds were validated and used for docking study to find the drug like molecules. The best molecules from the final set of screening candidates can be evaluated in cell lines and may represent a novel class of BCR-ABL inhibitors.
Abbreviations
CML - Chronic myeloid leukemia, PDGFR - Platelet derived growth factor receptor, TKI - Tyrosine kinase inhibitors.
doi:10.6026/97320630008658
PMCID: PMC3449370  PMID: 23055606
Ligand docking; BCR-ABL; Nilotinib; Glide score; Pharmacophore modeling
7.  Pristimerin induces apoptosis in imatinib-resistant chronic myelogenous leukemia cells harboring T315I mutation by blocking NF-κB signaling and depleting Bcr-Abl 
Molecular Cancer  2010;9:112.
Background
Chronic myelogenous leukemia (CML) is characterized by the chimeric tyrosine kinase Bcr-Abl. Bcr-Abl-T315I is the notorious point mutation that causes resistance to imatinib and the second generation tyrosine kinase inhibitors, leading to poor prognosis. CML blasts have constitutive p65 (RelA NF-κB) transcriptional activity, and NF-κB may be a potential target for molecular therapies in CML that may also be effective against CML cells with Bcr-Abl-T315I.
Results
In this report, we discovered that pristimerin, a quinonemethide triterpenoid isolated from Celastraceae and Hippocrateaceae, inhibited growth and induced apoptosis in CML cells, including the cells harboring Bcr-Abl-T315I mutation. Additionally, pristimerin inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Pristimerin blocked the TNFα-induced IκBα phosphorylation, translocation of p65, and expression of NF-κB-regulated genes. Pristimerin inhibited two steps in NF-κB signaling: TAK1→IKK and IKK→IκBα. Pristimerin potently inhibited two pairs of CML cell lines (KBM5 versus KBM5-T315I, 32D-Bcr-Abl versus 32D-Bcr-Abl-T315I) and primary cells from a CML patient with acquired resistance to imatinib. The mRNA and protein levels of Bcr-Abl in imatinib-sensitive (KBM5) or imatinib-resistant (KBM5-T315I) CML cells were reduced after pristimerin treatment. Further, inactivation of Bcr-Abl by imatinib pretreatment did not abrogate the TNFα-induced NF-κB activation while silencing p65 by siRNA did not affect the levels of Bcr-Abl, both results together indicating that NF-κB inactivation and Bcr-Abl inhibition may be parallel independent pathways.
Conclusion
To our knowledge, this is the first report to show that pristimerin is effective in vitro and in vivo against CML cells, including those with the T315I mutation. The mechanisms may involve inhibition of NF-κB and Bcr-Abl. We concluded that pristimerin could be a lead compound for further drug development to overcome imatinib resistance in CML patients.
doi:10.1186/1476-4598-9-112
PMCID: PMC2893099  PMID: 20482842
8.  Ponatinib Is a Pan-BCR-ABL Kinase Inhibitor: MD Simulations and SIE Study 
PLoS ONE  2013;8(11):e78556.
BCR-ABL kinase domain inhibition can be used to treat chronic myeloid leukemia. The inhibitors such as imatinib, dasatinib and nilotinib are effective drugs but are resistant to some BCR-ABL mutations. The pan-BCR-ABL kinase inhibitor ponatinib exhibits potent activity against native, T315I, and all other clinically relevant mutants, and showed better inhibition than the previously known inhibitors. We have studied the molecular dynamics simulations and calculated solvated interaction energies of native and fourteen mutant BCR-ABL kinases (M244V, G250E, Q252H, Y253F, Y253H, E255K, E255V, T315A, T315I, F317L, F317V, M351T, F359V and H396P) complexed with ponatinib. These studies revealed that the interactions between ponatinib and individual residues in BCR-ABL kinase are also affected due to the remote residue mutations. We report that some residues, Met244, Lys245, Gln252, Gly254, Leu370 and Leu298 do not undergo any conformational changes, while the fluctuations in residues from P-loop, β3-, β5- strands and αC- helix are mainly responsible for ponatinib binding to native and all mutant BCR-ABL kinases. Our work provides the molecular mechanisms of native and mutant BCR-ABL kinases inhibition by ponatinib at atomic level that has not been studied before.
doi:10.1371/journal.pone.0078556
PMCID: PMC3827254  PMID: 24236021
9.  Janus kinase 2 regulates Bcr–Abl signaling in chronic myeloid leukemia 
Leukemia  2010;25(3):463-472.
Despite the success of imatinib mesylate (IM) in the early chronic phase of chronic myeloid leukemia (CML), patients are resistant to IM and other kinase inhibitors in the later stages of CML. Our findings indicate that inhibition of Janus kinase 2 (Jak2) in Bcr–Abl+ cells overcomes IM resistance although the precise mechanism of Jak2 action is unknown. Knocking down Jak2 in Bcr–Abl+ cells reduced levels of the Bcr–Abl protein and also the phosphorylation of Tyr177 of Bcr–Abl, and Jak2 overexpression rescued these knockdown effects. Treatment of Bcr–Abl+ cells with Jak2 inhibitors for 4–6 h but not with IM also reduced Bcr–Abl protein and pTyr177 levels. In vitro kinase experiments performed with recombinant Jak2 showed that Jak2 readily phosphorylated Tyr177 of Bcr–Abl (a Jak2 consensus site, YvnV) whereas c-Abl did not. Importantly, Jak2 inhibition decreased pTyr177 Bcr–Abl in immune complexes but did not reduce levels of Bcr–Abl, suggesting that the reduction of Bcr–Abl by Jak2 inhibition is a separate event from phosphorylation of Tyr177. Jak2 inhibition by chemical inhibitors (TG101209/WP1193) and Jak2 knockdown diminished the activation of Ras, PI-3 kinase pathways and reduced levels of pTyrSTAT5. These findings suggest that Bcr–Abl stability and oncogenic signaling in CML cells are under the control of Jak2.
doi:10.1038/leu.2010.287
PMCID: PMC3072513  PMID: 21183952
chronic myeloid leukemia; Jak2; Bcr–Abl; Bcr–Abl ptyrosine 177; Jak2 inhibitors
10.  Triptolide induces cell death independent of cellular responses to imatinib in blast crisis CML cells including quiescent CD34+ primitive progenitor cells 
Molecular cancer therapeutics  2009;8(9):2509-2516.
The advent of Bcr-Abl tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of CML. However, resistance evolves due to BCR-ABL mutations and other mechanisms. Furthermore, patients with blast crisis (BC) CML are less responsive and quiescent CML stem cells are insensitive to these inhibitors. We found that triptolide, a diterpenoid, at nM concentrations, promoted equally significant death of KBM5 cells, a cell line derived from a Bcr-Abl-bearing BC CML patient and KBM5STI571 cells, an imatinib-resistant KBM5 subline bearing the T315I mutation. Similarly, Ba/F3 cells harboring mutated BCR-ABL were as sensitive as Ba/F3Bcr-Ablp210wt cells to triptolide. Importantly, triptolide induced apoptosis in primary samples from BC CML patients, who showed resistance to Bcr-Abl TKIs in vivo, with less toxicity to normal cells. Triptolide decreased XIAP, Mcl-1, and Bcr-Abl protein levels in K562, KBM5, KBM5STI571 cells and in cells from BC CML patients. It sensitized KBM5, but not KBM5STI571 cells to imatinib. More importantly, triptolide also induced death of quiescent CD34+ CML progenitor cells, a major problem in the therapy of CML with TKIs. Collectively, these results suggest that triptolide potently induces BC CML cell death independent of the cellular responses to Bcr-Abl TKIs, suggesting that triptolide could eradicate residual quiescent CML progenitor cells in TKI-treated patients and benefit TKI-resistant BC CML patients.
doi:10.1158/1535-7163.MCT-09-0386
PMCID: PMC2754862  PMID: 19723894
triptolide; XIAP; Mcl-1; Bcr-Abl; quiescent CD34+ CML cells
11.  A novel mechanism for Bcr-Abl action: Bcr-Abl-mediated induction of the eIF4F translation initiation complex and mRNA translation 
Oncogene  2006;26(8):1188-1200.
The oncogenic kinase Bcr-Abl is thought to cause chronic myelogenous leukemia (CML) by altering the transcription of specific genes with growth- and survival-promoting functions. Recently Bcr-Abl has also been shown to activate an important regulator of protein synthesis, the mammalian target of rapamycin (mTOR), which suggests that dysregulated translation may also contribute to CML pathogenesis. In this study, we found that both Bcr-Abl and the rapamycin-sensitive mTORC1 complex contribute to the phosphorylation (inactivation) of 4E-BP1, an inhibitor of the eIF4E translation initiation factor. Experiments with rapamycin and the Bcr-Abl inhibitor, imatinib mesylate, in Bcr-Abl-expressing cell lines and primary CML cells indicated that Bcr-Abl and mTORC1 induced formation of the translation initiation complex, eIF4F. This was characterized by reduced 4E-BP1- and increased eIF4G-binding to eIF4E, two events that lead to assembly of eIF4F. One target transcript is cyclin D3, which is regulated in Bcr-Abl-expressing cells by both Bcr-Abl and mTORC1 in a translational manner. In addition, the combination of imatinib and rapamycin was found to act synergistically against committed CML progenitors from chronic and blast phase patients. These experiments establish a novel mechanism of action for Bcr-Abl, and they provide insights into the modes of action of imatinib mesylate and rapamycin in treatment of CML. They also suggest that aberrant cap-dependent mRNA translation may be a therapeutic target in Bcr-Abl-driven malignancies.
doi:10.1038/sj.onc.1209901
PMCID: PMC2527622  PMID: 16936779
CML; eIF4F; mTOR; cap-dependent translation
12.  Allosteric inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated BCR-ABL and BCR-ABL-T315I 
BMC Cancer  2012;12:411.
Background
Chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphatic leukemia (Ph + ALL) are caused by the t(9;22), which fuses BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase “escapes” the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs) Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the ‘gatekeeper’ mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia.
Methods
The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC) from Ph + ALL-patients.
Results
Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner.
Conclusions
Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors.
doi:10.1186/1471-2407-12-411
PMCID: PMC3488316  PMID: 22985168
Philadelphia chromosome; BCR/ABL; “gatekeeper” mutation T315I; Allosteric inhibition; Abl kinase inhibitors; Molecular therapy
13.  The effect of the dual Src/Abl kinase inhibitor AZD0530 on Philadelphia positive leukaemia cell lines 
BMC Cancer  2009;9:53.
Background
Imatinib mesylate, a selective inhibitor of Abl tyrosine kinase, is efficacious in treating chronic myeloid leukaemia (CML) and Ph+ acute lymphoblastic leukaemia (ALL). However, most advanced-phase CML and Ph+ ALL patients relapse on Imatinib therapy. Several mechanisms of refractoriness have been reported, including the activation of the Src-family kinases (SFK). Here, we investigated the biological effect of the new specific dual Src/Abl kinase inhibitor AZD0530 on Ph+ leukaemic cells.
Methods
Cell lines used included BV173 (CML in myeloid blast crisis), SEM t(4;11), Ba/F3 (IL-3 dependent murine pro B), p185Bcr-Abl infected Ba/F3 cells, p185Bcr-Abl mutant infected Ba/F3 cells, SupB15 (Ph+ ALL) and Imatinib resistant SupB15 (RTSupB15) (Ph+ ALL) cells. Cells were exposed to AZD0530 and Imatinib. Cell proliferation, apoptosis, survival and signalling pathways were assessed by dye exclusion, flow cytometry and Western blotting respectively.
Results
AZD0530 specifically inhibited the growth of, and induced apoptosis in CML and Ph+ ALL cells in a dose dependent manner, but showed only marginal effects on Ph- ALL cells. Resistance to Imatinib due to the mutation Y253F in p185Bcr-Abl was overcome by AZD0530. Combination of AZD0530 and Imatinib showed an additive inhibitory effect on the proliferation of CML BV173 cells but not on Ph+ ALL SupB15 cells. An ongoing transphosphorylation was demonstrated between SFKs and Bcr-Abl. AZD0530 significantly down-regulated the activation of survival signalling pathways in Ph+ cells, resistant or sensitive to Imatinib, with the exception of the RTSupB15.
Conclusion
Our results indicate that AZD0530 targets both Src and Bcr-Abl kinase activity and reduces the leukaemic maintenance by Bcr-Abl.
doi:10.1186/1471-2407-9-53
PMCID: PMC2654659  PMID: 19216789
14.  Expression and Activity of Fyn Mediate Proliferation and Blastic Features of Chronic Myelogenous Leukemia 
PLoS ONE  2012;7(12):e51611.
The BCR-ABL1 oncogene is a tyrosine kinase that activates many signaling pathways, resulting in the induction of chronic myeloid leukemia (CML). Kinase inhibitors, such as imatinib, have been developed for the treatment of CML; however, the terminal, blast crisis phase of the disease remains a clinical challenge. Blast crisis CML is difficult to treat due to resistance to tyrosine kinase inhibitors, increased genomic instability and acquired secondary mutations. Our recent studies uncovered a role for Fyn in promoting BCR-ABL1 mediated cell growth and sensitivity to imatinib. Here we demonstrate that Fyn contributes to BCR-ABL1 induced genomic instability, a feature of blast crisis CML. Bone marrow cells and mouse embryonic fibroblasts derived from Fyn knockout mice transduced with BCR-ABL1 display slowed growth and clonogenic potential as compared to Fyn wild-type BCR-ABL1 expressing counterparts. K562 cells overexpressing constitutively active Fyn kinase were larger in size and displayed an accumulation of genomic abnormalities such as chromosomal aberrations and polyploidy. Importantly, loss of Fyn protected mouse embryonic fibroblast cells from increased number of chromosomal aberrations and fragments induced by BCR-ABL1. Together, these results reveal a novel role for Fyn in regulating events required for genomic maintenance and suggest that Fyn kinase activity plays a role in the progression of CML to blast crisis.
doi:10.1371/journal.pone.0051611
PMCID: PMC3524192  PMID: 23284724
15.  Interplay between Kinase Domain Autophosphorylation and F-Actin Binding Domain in Regulating Imatinib Sensitivity and Nuclear Import of BCR-ABL 
PLoS ONE  2011;6(2):e17020.
Background
The constitutively activated BCR-ABL tyrosine kinase of chronic myeloid leukemia (CML) is localized exclusively to the cytoplasm despite the three nuclear localization signals (NLS) in the ABL portion of this fusion protein. The NLS function of BCR-ABL is re-activated by a kinase inhibitor, imatinib, and in a kinase-defective BCR-ABL mutant. The mechanism of this kinase-dependent inhibition of the NLS function is not understood.
Methodology/Principal Findings
By examining the subcellular localization of mutant BCR-ABL proteins under conditions of imatinib and/or leptomycin B treatment to inhibit nuclear export, we have found that mutations of three specific tyrosines (Y232, Y253, Y257, according to ABL-1a numbering) in the kinase domain can inhibit the NLS function of kinase-proficient and kinase-defective BCR-ABL. Interestingly, binding of imatinib to the kinase-defective tyrosine-mutant restored the NLS function, suggesting that the kinase domain conformation induced by imatinib-binding is critical to the re-activation of the NLS function. The C-terminal region of ABL contains an F-actin binding domain (FABD). We examined the subcellular localization of several FABD-mutants and found that this domain is also required for the activated kinase to inhibit the NLS function; however, the binding to F-actin per se is not important. Furthermore, we found that some of the C-terminal deletions reduced the kinase sensitivity to imatinib.
Conclusions/Significance
Results from this study suggest that an autophosphorylation-dependent kinase conformation together with the C-terminal region including the FABD imposes a blockade of the BCR-ABL NLS function. Conversely, conformation of the C-terminal region including the FABD can influence the binding affinity of imatinib for the kinase domain. Elucidating the structural interactions among the kinase domain, the NLS region and the FABD may therefore provide insights on the design of next generation BCR-ABL inhibitors for the treatment of CML.
doi:10.1371/journal.pone.0017020
PMCID: PMC3037956  PMID: 21347248
16.  Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl 
Bcr/Abl is a chimeric oncogene that can cause both acute and chronic human leukemias. Bcr/Abl-encoded proteins exhibit elevated kinase activity compared to c-Abl, but the mechanisms of transformation are largely unknown. Some of the biological effects of Bcr/Abl overlap with those of hematopoietic cytokines, particularly interleukin 3 (IL-3). Such effects include mitogenesis, enhanced survival, and enhanced basophilic differentiation. Therefore, it has been suggested that p210Bcr/Abl and the IL-3 receptor may activate some common signal transduction pathways. An important pathway for IL-3 signaling involves activation of the Janus family kinases (JAKs) and subsequent tyrosyl phosphorylation of STAT proteins (signal transducers and activators of transcription). This pathway directly links growth factor receptors to gene transcription. We analyzed JAK activation, STAT protein phosphorylation, and the formation of specific DNA-binding complexes containing STAT proteins, in a series of leukemia cell lines transformed by Bcr/Abl or other oncogenes. We also examined these events in cell lines transformed by a temperature sensitive (ts) mutant of Bcr/Abl, where the kinase activity of Abl could be regulated. STAT1 and STAT5 were found to be constitutively phosphorylated in 32D, Ba/F3, and TF-1 cells transformed by Bcr/Abl, but not in the untransformed parental cell lines in the absence of IL-3. Phosphorylation of STAT1 and STAT5 was also observed in the human leukemia cell lines K562 and BV173, which express the Bcr/Abl oncogene, but not in several Bcr/Abl- negative leukemia cell lines. Phosphorylation of STAT1 and STAT5 was directly due to the tyrosine kinase activity of Bcr/Abl since it could be activated or deactivated by temperature shifting of cells expressing the Bcr/Abl ts mutant. DNA-STAT complexes were detected in all Bcr/Abl- transformed cell lines and they were supershifted by antibodies against STAT1 and STAT5. DNA-STAT complexes in 32Dp210Bcr/Abl cells were similar, but not identical, to those formed after IL-3 stimulation. It is interesting to note that JAK kinases (JAK1, JAK2, JAK3, and Tyk2) were not consistently activated in Bcr/Abl-positive cells. These data suggest that STATs can be activated directly by Bcr/Abl, possibly bypassing JAK family kinase activation. Overall, our results suggest a novel mechanism that could contribute to some of the major biological effects of Bcr/Abl transformation.
PMCID: PMC2192351  PMID: 8642285
17.  Src-family kinases in the development and therapy of Philadelphia chromosome-positive chronic myeloid leukemia and acute lymphoblastic leukemia 
Leukemia & Lymphoma  2008;49(1):19-26.
The BCR-ABL kinase inhibitor imatinib has shown significant efficacy in chronic myeloid leukemia (CML) and is the standard front-line therapy for patients in chronic phase. However, a substantial number of patients are either primarily refractory or acquire resistance to imatinib. While a number of mechanisms are known to confer resistance to imatinib, increasing evidence has demonstrated a role for BCR-ABL–independent pathways. The Src-family kinases (SFKs) are one such pathway and have been implicated in imatinib resistance. Additionally, these kinases are key to the progression of CML and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). The dual SFK/BCR-ABL inhibitor dasatinib is now clinically available and has markedly greater potency compared with imatinib against native BCR-ABL and the majority of imatinib resistant BCR-ABL mutants. Therefore, this agent, as well as other dual SFK/BCR-ABL inhibitors under development, could provide added therapeutic advantages by overcoming both BCR-ABL– dependent (i.e., BCR-ABL mutations) and – independent forms of imatinib resistance and delaying transition to advanced phase disease. In this review, we discuss the preclinical and clinical evidence demonstrating the involvement of SFKs in imatinib resistance and the progression of CML and Ph+ ALL, as well as the potential role of dual SFK/BCR-ABL inhibition in the management of these diseases.
doi:10.1080/10428190701713689
PMCID: PMC2430171  PMID: 18203007
Src; leukemia; BCR-ABL; dasatinib; imatinib resistant
18.  Nilotinib: optimal therapy for patients with chronic myeloid leukemia and resistance or intolerance to imatinib 
Chronic myeloid leukemia (CML) is the consequence of a single balanced translocation that produces the BCR-ABL fusion oncogene which is detectable in over 90% of patients at presentation. The BCR-ABL inhibitor imatinib mesylate (IM) has improved survival in all phases of CML and is the standard of care for newly diagnosed patients in chronic phase. Despite the very significant therapeutic benefits of IM, a small minority of patients with early stage disease do not benefit optimally while IM therapy in patients with advanced disease is of modest benefit in many. Diverse mechanisms may be responsible for IM failures, with point mutations within the Bcr-Abl kinase domain being amongst the most common resistance mechanisms described in patients with advanced CML. The development of novel agents designed to overcome IM resistance, while still primarily targeted on BCR-ABL, led to the creation of the high affinity aminopyrimidine inhibitor, nilotinib. Nilotinib is much more potent as a BCR-ABL inhibitor than IM and inhibits both wild type and IM-resistant BCR-ABL with significant clinical activity across the entire spectrum of BCR-ABL mutants with the exception of T315I. The selection of a second generation tyrosine kinase inhibitor to rescue patients with imatinib failure will be based on several factors including age, co-morbid medical problems and ABL kinase mutational profile. It should be noted that while the use of targeted BCR-ABL kinase inhibitors in CML represents a paradigm shift in CML management these agents are not likely to have activity against the quiescent CML stem cell pool. The purpose of this review is to summarize the pre-clinical and clinical data on nilotinib in patients with CML who have failed prior therapy with IM or dasatinib.
PMCID: PMC2769239  PMID: 19920925
nilotinib; chronic myeloid leukemia; imatinib
19.  Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing 
BMC Cancer  2015;15:45.
Background
The evolution of mutations in the BCR-ABL1 fusion gene transcript renders CML patients resistant to tyrosine kinase inhibitor (TKI) based therapy. Thus screening for BCR-ABL1 mutations is recommended particularly in patients experiencing poor response to treatment. Herein we describe a novel approach for the detection and surveillance of BCR-ABL1 mutations in CML patients.
Methods
To detect mutations in the BCR-ABL1 transcript we developed an assay based on the Pacific Biosciences (PacBio) sequencing technology, which allows for single-molecule long-read sequencing of BCR-ABL1 fusion transcript molecules. Samples from six patients with poor response to therapy were analyzed both at diagnosis and follow-up. cDNA was generated from total RNA and a 1,6 kb fragment encompassing the BCR-ABL1 transcript was amplified using long range PCR. To estimate the sensitivity of the assay, a serial dilution experiment was performed.
Results
Over 10,000 full-length BCR-ABL1 sequences were obtained for all samples studied. Through the serial dilution analysis, mutations in CML patient samples could be detected down to a level of at least 1%. Notably, the assay was determined to be sufficiently sensitive even in patients harboring a low abundance of BCR-ABL1 levels. The PacBio sequencing successfully identified all mutations seen by standard methods. Importantly, we identified several mutations that escaped detection by the clinical routine analysis. Resistance mutations were found in all but one of the patients. Due to the long reads afforded by PacBio sequencing, compound mutations present in the same molecule were readily distinguished from independent alterations arising in different molecules. Moreover, several transcript isoforms of the BCR-ABL1 transcript were identified in two of the CML patients. Finally, our assay allowed for a quick turn around time allowing samples to be reported upon within 2 days.
Conclusions
In summary the PacBio sequencing assay can be applied to detect BCR-ABL1 resistance mutations in both diagnostic and follow-up CML patient samples using a simple protocol applicable to routine diagnosis. The method besides its sensitivity, gives a complete view of the clonal distribution of mutations, which is of importance when making therapy decisions.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1046-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-015-1046-y
PMCID: PMC4335374
20.  Disrupting BCR-ABL in Combination with Secondary Leukemia-Specific Pathways in CML Cells Leads to Enhanced Apoptosis and Decreased Proliferation 
Molecular pharmaceutics  2012;10(1):270-277.
Chronic myeloid leukemia (CML) is a myeloproliferative disorder caused by expression of the fusion gene BCR-ABL following a chromosomal translocation in the hematopoietic stem cell.1 Therapeutic management of CML uses tyrosine kinase inhibitors (TKIs), which blocks ABL-signaling and effectively kill peripheral cells with BCR-ABL. However, TKIs are not curative, and chronic use of is required in order to treat CML. The primary failure for TKIs is through development of a resistant population due to mutations in the TKI binding regions.2, 3 This led us to develop the mutant coiled-coil, CCmut2, an alternative method for BCR-ABL signaling inhibition by targeting the N-terminal oligomerization domain of BCR, necessary for ABL activation.4 In this report we explore additional pathways which are important for leukemic stem cell survival in K562 cells. Using a candidate-based approach we test the combination of CCmut2 and inhibitors of unique secondary pathways in leukemic cells. Transformative potential was reduced following silencing of the leukemic stem cell factor Alox55 by RNA interference. Furthermore, blockade of the oncogenic protein MUC-16 by the novel peptide GO-201 yielded reductions in proliferation and increased cell death. Finally, we found that inhibiting macroautophagy7 using chloroquine in addition to blocking BCR-ABL signaling with the CCmut2 was most effective in limiting cell survival and proliferation. This study has elucidated possible combination therapies for CML using novel blockade of BCR-ABL and secondary leukemia-specific pathways.
doi:10.1021/mp300405n
PMCID: PMC3604893  PMID: 23211037
CML; Coiled-Coil; CCmut2; zileuton; GO-210; chloroquine; combination therapy; K562; BCR-ABL
21.  Contribution of BCR-ABL kinase domain mutations to imatinib mesylate resistance in Philadelphia chromosome positive Malaysian chronic myeloid leukemia patients 
Hematology Reports  2012;4(4):e23.
Development of resistance to imatinib mesylate (IM) in chronic myeloid leukemia (CML) patients is mediated by different mechanisms that can be classified as BCR-ABL dependent or BCR-ABL independent pathways. BCR-ABL dependent mechanisms are most frequently associated with point mutations in tyrosine kinase domain (TKD) of BCR-ABL1 and also with BCR-ABL gene amplification. Many different types and frequencies of mutations have been reported in different studies, probably due to the different composition of study cohorts. Since no reports are available from Malaysia, this study was undertaken to investigate the frequency and pattern of BCR-ABL kinase domain mutations using dHPLC followed by sequencing, and also status of BCR-ABL gene amplification using fluorescence in situ hybridization (FISH) on 40 IM resistant Malaysian CML patients. Mutations were detected in 13 patients (32.5%). Five different types of mutations (T315I, E255K, Y253H, M351T, V289F) were identified in these patients. In the remaining 27 IM resistant CML patients, we investigated the contribution made by BCR-ABL gene amplification, but none of these patients showed amplification. It is presumed that the mechanisms of resistance in these 27 patients might be due to BCR-ABL independent pathways. Different mutations confer different levels of resistance and, therefore, detection and characterization of TKD mutations is highly important in order to guide therapy in CML patients.
doi:10.4081/hr.2012.e23
PMCID: PMC3555211  PMID: 23355941
chronic myeloid leukemia; imatinib mesylate; BCR-ABL dependent mechanisms; tyrosine kinase domain; mutation.
22.  Association Between Imatinib-Resistant BCR-ABL Mutation-Negative Leukemia and Persistent Activation of LYN Kinase 
Background
Imatinib is a tyrosine kinase inhibitor that is used to treat chronic myelogenous leukemia (CML). BCR-ABL mutations are associated with failure of imatinib treatment in many CML patients. LYN kinase regulates survival and responsiveness of CML cells to inhibition of BCR-ABL kinase, and differences in LYN regulation have been found between imatinib-sensitive and -resistant CML cell lines.
Methods
We evaluated cells from 12 imatinib-resistant CML patients with mutation-negative BCR-ABL and from six imatinib-sensitive patients who discontinued therapy because of imatinib intolerance. Phosphorylation of BCR-ABL and LYN was assessed in patient cells and cell lines by immunoblotting with activation state–specific antibodies, co-immunoprecipitation studies, and mass spectroscopy analysis of phosphopeptides. Cell viability, caspase activation, and apoptosis were also measured. Mutations were analyzed by sequencing. The effect of silencing LYN with short interfering RNAs (siRNAs) or reducing activation by treatment with tyrosine kinase inhibitors was evaluated in cell lines and patient cells.
Results
Imatinib treatment suppressed LYN phosphorylation in cells from imatinib-sensitive CML patients and imatinib-sensitive cell lines. Imatinib treatment blocked BCR-ABL signaling but did not suppress LYN phosphorylation in cells from imatinib-resistant patients, and persistent activation of LYN kinase was not associated with mutations in LYN kinase or its carboxyl-terminal regulatory domains. Unique LYN phosphorylation sites (tyrosine-193 and tyrosine-459) and associated proteins (c-Cbl and p80) were identified in cells from imatinib-resistant patients. Reducing LYN expression (siRNA) or activation (dasatinib) was associated with loss of cell survival and cytogenetic or complete hematologic responses in imatinib-resistant disease.
Conclusions
LYN activation was independent of BCR-ABL in cells from imatinib-resistant patients. Thus, LYN kinase may be involved in imatinib resistance in CML patients with mutation-negative BCR-ABL and its direct inhibition is consistent with clinical responses in these patients.
doi:10.1093/jnci/djn188
PMCID: PMC2902818  PMID: 18577747
23.  Global Phosphoproteomics Reveals Crosstalk between Bcr-Abl and Negative Feedback Mechanisms Controlling Src Signaling 
Science signaling  2011;4(166):ra18.
In subtypes and late stages of leukemias driven by the tyrosine kinase fusion protein Bcr-Abl, Src signaling critically contributes to the leukemic phenotype. We performed global tyrosine phosphoprofiling using quantitative mass spectrometry of Bcr-Abl transformed cells in which the activities of the Src family kinases (SFKs) were perturbed to build a detailed context-dependent network of cancer signaling. Perturbation of the SFKs Lyn and Hck with genetics or inhibitors revealed Bcr-Abl downstream phosphorylation events either mediated by or independent of SFKs. We identified multiple negative feedback mechanisms within the network of signaling events affected by Bcr-Abl and SFKs, and found that Bcr-Abl attenuated these inhibitory mechanisms. The Csk binding protein Pag1 (also known as Cbp) and the tyrosine phosphatase Ptpn18 both mediated negative feedback to SFKs. We observed Bcr-Abl-mediated phosphorylation of the phosphatase Shp2 (Ptpn11) and this may contribute to the suppression of these negative feedback mechanisms to promote Bcr-Abl-activated SFK signaling. Csk and a kinase-deficient Csk mutant both produced similar globally repressive signaling consequences, suggesting a critical role for the adaptor protein function of Csk in its inhibition of Bcr-Abl and SFK signaling. The identified Bcr-Abl-activated SFK regulatory mechanisms are candidates for dysregulation during leukemia progression and acquisition of SFK-mediated drug resistance.
doi:10.1126/scisignal.2001314
PMCID: PMC4057100  PMID: 21447799
24.  BCR/ABL Directly Inhibits Expression of SHIP, an SH2-Containing Polyinositol-5-Phosphatase Involved in the Regulation of Hematopoiesis 
Molecular and Cellular Biology  1999;19(11):7473-7480.
The BCR/ABL oncogene causes chronic myelogenous leukemia (CML), a myeloproliferative disorder characterized by clonal expansion of hematopoietic progenitor cells and granulocyte lineage cells. The SH2-containing inositol-5-phosphatase SHIP is a 145-kDa protein which has been shown to regulate hematopoiesis in mice. Targeted disruption of the murine SHIP gene results in a myeloproliferative syndrome characterized by a dramatic increase in numbers of granulocyte-macrophage progenitor cells in the marrow and spleen. Also, hematopoietic progenitor cells from SHIP−/− mice are hyperresponsive to certain hematopoietic growth factors, a phenotype very similar to the effects of BCR/ABL in murine cells. In a series of BCR/ABL-transformed hematopoietic cell lines, Philadelphia chromosome (Ph)-positive cell lines, and primary cells from patients with CML, the expression of SHIP was found to be absent or substantially reduced compared to untransformed cell lines or leukemia cells lacking BCR/ABL. Ba/F3 cells in which expression of BCR/ABL was under the control of a tetracycline-inducible promoter showed rapid loss of p145 SHIP, coincident with induction of BCR/ABL expression. Also, an ABL-specific tyrosine kinase inhibitor, CGP57148B (STI571), rapidly caused reexpression of SHIP, indicating that BCR/ABL directly, but reversibly, regulates the expression of SHIP protein. The estimated half-life of SHIP protein was reduced from 18 h to less than 3 h. However, SHIP mRNA also decreased in response to BCR/ABL, suggesting that SHIP protein levels could be affected by more than one mechanism. Reexpression of SHIP in BCR/ABL-transformed Ba/F3 cells altered the biological behavior of cells in culture. The reduction of SHIP due to BCR/ABL is likely to directly contribute to the pathogenesis of CML.
PMCID: PMC84744  PMID: 10523635
25.  Tyrosine kinase inhibitor AG1024 exerts antileukaemic effects on STI571-resistant Bcr-Abl expressing cells and decreases AKT phosphorylation 
British Journal of Cancer  2004;91(9):1735-1741.
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.
doi:10.1038/sj.bjc.6602190
PMCID: PMC2409959  PMID: 15494718
Bcr-Abl; Tyrphostin AG1024; AKT; IGF1; tyrosine kinase; DNA-PKcs

Results 1-25 (647096)