A Study of the Binding Mode and the In Vitro Activity of the Protein Tyrosine Kinase Inhibitor SKI-606 in the BCR-ABL Positive Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2335-2335
Author(s):  
Tiziana Grafone ◽  
Simona Soverini ◽  
Gianluca Tasco ◽  
Manuela Mancini ◽  
Frank Boschelli ◽  
...  
Keyword(s):  
Protein Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
K562 Cells ◽  
Binding Mode ◽  
Inactive Conformation ◽  
Abl Kinase ◽  
Cd34 Cells

Abstract Resistance to the BCR-ABL inhibitor imatinib mesylate (IM) is often caused by the selection of leukemic clones harboring mutations that destabilize the inactive conformation of BCR-ABL, to which IM preferentially binds, shifting the equilibrium toward the active kinase conformation. Hence the need for second -generation kinase inhibitors with a greater flexibility in binding to different BCR-ABL conformations. The 4-anilino-3 quinolinecarbonitrile SKI-606 is a novel Src and ABL kinase inhibitor. We report here that SKI-606 is a potent and antiproliferative agent when tested in K562 cell line and CD34+ cells from patients with chronic myeloid leukemia (CML) blast crisis. In K562 cells, SKI-606 treatment caused a dose-dependent decrease in cell viability accompanied by accumulation in subG1 phase, an effect not observed in BCR-ABL-negative HL-60 cells. Treatment of K562 with 100 nM SKI-606 for 24 hours caused a complete dephosphorylation of Lyn, Stat5 and Bcl-xl, while AKT and Bad phosphorylation was only diminished. Unexpectedly, the phosphorylation of BCR-ABL was less affected. SKI-606 treatment caused a shift to the subG1 phase also of CD34+ cells isolated from both IM-sensitive and resistant patients, the latter harboring the BCR-ABL mutations F359V, Y253H, E255V and E255K. The inhibitory concentration 50% (IC50) was 100 nM SKI-606 for Y253H, E255V, E255K and 1000nM for F359V. Cytofluorimetric analysis of cells from IM- sensitive CML patients indicated an accumulation in subG1 phase following treatment with SKI-606 alone or in combination with IM. Because the crystal structure of the BCR-ABL kinase domain in complex with SKI-606 has not yet been determined and the mode of binding of this inhibitor is unknown, we first used a molecular docking approach to determine SKI-606 binding mode to wild type (wt) form of the BCR-ABL kinase. We found that the interaction between SKI-606 and BCR-ABL was more stable when the activation loop was in the inactive conformation. Moreover, we found that SKI-606 retained the ability of efficiently binding all the above mentioned BCR-ABL variants, but not the T315I. Finally, we identified six BCR-ABL residues located around SKI-606 that, if mutated, could potentially be able to interfere with the SKI-606/BCR-ABL interaction: the charged residues K271, D381 and H361; the hydrophobic/aliphatic residues V299, A380 and M318. These data help refining the use of SKI-606 for treatment of BCR-ABL positive leukemias.

Inhibition of Bcl-2/Bcl-XL Promotes Apoptosis in Blast Crisis CML Including Quiescent Primitive Progenitor Cells Regardless of Cellular Responses to Tyrosine Kinase Inhibitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 646-646
Author(s):  
Duncan H. Mak ◽  
Wendy D. Schober ◽  
Marina Konopleva ◽  
Jorge Cortes ◽  
Hagop M. Kantarjian ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Progenitor Cells ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Antiapoptotic Proteins ◽  
Cell Compartments ◽  
Cd34 Cells

Abstract Abstract 646 The advent of imatinib, a Bcr-Abl tyrosine kinase inhibitor revolutionized the treatment for patients with CML. Development of resistance, limited activity in blast crisis CML, and more importantly, insensitivity of quiescent primitive CD34+ CML progenitor cells are evolving problems facing this therapy. Antiapoptotic Bcl-2 proteins were known to be highly expressed in Bcr-Abl expressing cells and inhibition of Bcl-2/Bcl-XL by the selective inhibitor ABT-737 was reported to augment the killing of tyrosine kinase inhibitors in CML cells. However, its effect on quiescent primitive CD34+ CML progenitor cells is unknown. To investigate the effect of activating the apoptotic machinery in quiescent primitive CD34+CML progenitor cells, which are resistant to current therapies, we first compared the expression of antiapoptotic proteins in proliferating and quiescent primitive CD34+CML progenitor cells. Cells obtained from patients with blast crisis CML were stained with the fluorescent 5-(and 6-) carboxy-fluorescein diacetate succinimidyl ester, a cell proliferation tracking dye, and cultured in vitro for 4-6 days. Cells were then stained with CD34 antibody and FACS sorted into proliferating and quiescent CD34+/PI- CML progenitor cells. RNA levels of antiapoptotic proteins in these two cell populations (n=8) were determined by real-time RT-PCR: quiescent and proliferating primitive CD34+ CML progenitor cells expressed similar levels of Bcl-2, Bcl-XL, Mcl-1, and XIAP implying that like total blast cells, quiescent primitive CD34+CML progenitor cells may also be sensitive to agents targeting these proteins. We next treated 5 samples obtained from patients with blast crisis CML with ABT-737 and measured apoptosis in total CD34+ cells, proliferating CD34+ cells, and quiescent CD34+ cells. All 5 patients were resistant to or relapsed from imatinib and nilotinib and/or dasatinib treatments and they were insensitive to imatinib in vitro as expected. However, cells from 4 patients were sensitive to ABT-737, in bulk blasts and in both proliferating and quiescent CD34+ CML cell compartments: % specific apoptosis with 100 nM of ABT-737=40.8±7.7, 38.4±8.5, 40.0±5.1, respectively at 24 hours. Interestingly, when ABT-737 was combined with imatinib, cell death was greatly enhanced in cells from all 5 patients in all cell compartments (combination index=0.059±0.032, 0.041±0.025, 0.111±0.042, respectively). Furthermore, we showed previously, that triptolide, an antitumor agent from a Chinese herb, induces apoptosis in both proliferating and quiescent primitive CD34+CML progenitor cells by decreasing Mcl-1 which is a resistant factor for ABT-737, XIAP, and Bcr-Abl protein levels (Mak D. et al., MCT in press). When ABT-737 was combined with triptolide, a significant increase of cell death was found in total CD34+ and proliferating as well as quiescent primitive CD34+CML cells with combination index at EC50=0.57, 0.55, and 0.56, respectively in cells from the 5 patients suggesting a high degree of synergism. In summary, Bcl-2, Bcl-XL, Mcl-1, and XIAP are equally expressed in proliferating and quiescent primitive CML cells and targeting Bcl-2/Bcl-XL promotes death of blast crisis CML cells, tyrosine kinase inhibitor resistant CML cells, and quiescent primitive CD34+ CML progenitor cells. Researches suggest that the combination of apoptosis inducing agents and tyrosine kinase inhibitor is a novel strategy to overcome tyrosine kinase resistance, eradicate quiescent primitive CML progenitor cells, and improve current therapy for patients with CML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Leukemogenesis induced by wild-type and STI571-resistant BCR/ABL is potently suppressed by C/EBPα

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1353-1362 ◽  
Author(s):  
Giovanna Ferrari-Amorotti ◽  
Karen Keeshan ◽  
Michela Zattoni ◽  
Clara Guerzoni ◽  
Giorgio Iotti ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Drug Binding ◽  
Myelogenous Leukemia ◽  
Abl Kinase ◽  
Kinase Domain Mutation

Abstract Chronic phase–to–blast crisis transition in chronic myelogenous leukemia (CML) is associated with differentiation arrest and down-regulation of C/EBPα, a transcription factor essential for granulocyte differentiation. Patients with CML in blast crisis (CML-BC) became rapidly resistant to therapy with the breakpoint cluster region–Abelson murine leukemia (BCR/ABL) kinase inhibitor imatinib (STI571) because of mutations in the kinase domain that interfere with drug binding. We show here that the restoration of C/EBPα activity in STI571-sensitive or -resistant 32D-BCR/ABL cells induced granulocyte differentiation, inhibited proliferation in vitro and in mice, and suppressed leukemogenesis. Moreover, activation of C/EBPα eradicated leukemia in 4 of 10 and in 6 of 7 mice injected with STI571-sensitive or -resistant 32D-BCR/ABL cells, respectively. Differentiation induction and proliferation inhibition were required for optimal suppression of leukemogenesis, as indicated by the effects of p42 C/EBPα, which were more potent than those of K298E C/EBPα, a mutant defective in DNA binding and transcription activation that failed to induce granulocyte differentiation. Activation of C/EBPα in blast cells from 4 patients with CML-BC, including one resistant to STI571 and BMS-354825 and carrying the T315I Abl kinase domain mutation, also induced granulocyte differentiation. Thus, these data indicate that C/EBPα has potent antileukemia effects even in cells resistant to ATP-binding competitive tyrosine kinase inhibitors, and they portend the development of anti-leukemia therapies that rely on C/EBPα activation.

scholarly journals Design, Synthesis and Biological Evaluation of Pentacyclic Triterpene Derivatives: Optimization of Anti-ABL Kinase Activity

Molecules ◽  
2019 ◽  
Vol 24 (19) ◽  
pp. 3535 ◽  
Author(s):  
Halil I. Ciftci ◽  
Mohamed O. Radwan ◽  
Safiye E. Ozturk ◽  
N. Gokce Ulusoy ◽  
Ece Sozer ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
K562 Cells ◽  
Biological Evaluation ◽  
Benzyl Ester ◽  
Myelogenous Leukemia ◽  
Pentacyclic Triterpene ◽  
Abl Kinase ◽  
Molecular Targeted Drug ◽  
Benzyl Esters

Imatinib, an Abelson (ABL) tyrosine kinase inhibitor, is a lead molecular-targeted drug against chronic myelogenous leukemia (CML). To overcome its resistance and adverse effects, new inhibitors of ABL kinase are needed. Our previous study showed that the benzyl ester of gypsogenin (1c), a pentacyclic triterpene, has anti-ABL kinase and a subsequent anti-CML activity. To optimize its activities, benzyl esters of carefully selected triterpenes (PT1–PT6), from different classes comprising oleanane, ursane and lupane, and new substituted benzyl esters of gypsogenin (GP1–GP5) were synthesized. All of the synthesized compounds were purified and charachterized by different spectroscopic methods. Cytotoxicity of the parent triterpenes and the synthesized compounds against CML cell line K562 was examined; revealing three promising compounds PT5, GP2 and GP5 (IC50 5.46, 4.78 and 3.19 μM, respectively). These compounds were shown to inhibit extracellular signal-regulated kinase (ERK) downstream signaling, and induce apoptosis in K562 cells. Among them, PT5 was identified to have in vitro activity (IC50 = 1.44 μM) against ABL1 kinase, about sixfold of 1c, which was justified by molecular docking. The in vitro activities of GP2 and GP5 are less than PT5, hence they were supposed to possess other more mechanisms of cytotoxicity. In general, our design and derivatizations resulted in enhancing the activity against ABL1 kinase and CML cells.

Properties of CD34+ CML Cells That Predict Clinical Response to Tyrosine Kinase Inhibitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 324-324
Author(s):  
Xiaoyan Jiang ◽  
Donna Forrest ◽  
Franck Nicolini ◽  
Karen Lambie ◽  
Kyi Min Saw ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Cd34 Cells ◽  
Clinical Responses ◽  
Kinase Domain Mutations

Abstract Imatinib (IM) treatment causes remission in a majority of patients with chronic myeloid leukemia (CML) but relapses remain a problem. The frequent presence in relapsing cells of BCR-ABL kinase domain mutations suggests that their prior but undetected acquisition by rare CML stem cells may be a major contributor to IM treatment failures. We have recently demonstrated that enriched populations of CML stem cells (lin−CD34+CD38− cells) are relatively insensitive to IM and possess multiple unique features that would be expected to promote both innate and acquired mechanisms of resistance to BCR-ABL-targeted therapeutics. These include elevated BCR-ABL expression and tyrosine kinase activity, increased expression of ABCB1/MDR1 and ABCG2, decreased expression of OCT1, and a high degree of genetic instability, as demonstrated by a rapid accumulation of BCR-ABL mutations in vitro. To determine whether these parameters may be predictive of clinical responses to IM, immunomagnetically selected CD34+ stem/progenitor cells from 18 chronic phase CML patients’ samples obtained prior to IM therapy were evaluated and the results compared with subsequent clinical responses. Direct sequencing of transcripts cloned from extracts of freshly isolated CD34+ cells (10 clones/sample) detected a high frequency of pre-existing BCR-ABL kinase mutations in the CD34+ cells from 12 of 12 patients regardless of their subsequent IM responses (20–80%). Interestingly, a higher incidence of BCR-ABL kinase domain mutations was found in 5 IM-nonresponders (33–80% of transcripts showed ≥1 BCR-ABL kinase domain mutation) as compared to 5 IM-responders (values of 20-30%, P<0.02). A higher frequency of BCR-ABL kinase domain mutations was also detected in extracts of colonies generated from assays of cells harvested from 3-week suspension cultures initiated with the same starting CD34+ CML cells (21–68% vs 10–43%). A high incidence of BCR-ABL kinase domain mutations was also documented in freshly isolated or cultured CD34+ cells from 2 patients who developed sudden blast crisis (50–63% and 17–83%). Overall, 38 different mutations were identified from freshly isolated CD34+ CML cells and >50 additional mutations were identified in the progeny of CD34+ CML cells cultured ± IM. These included 15 point mutations frequently associated with clinical IM resistance (including G250, Q252, E255, T315, M351, F359 and H396) and >40 mutations not previously described. Furthermore, freshly isolated CD34+ cells from IM-nonresponders (including the 2 patients who developed blast crisis, n=10) showed a greater resistance to IM in vitro (∼2 fold, P< 0.001 with 5 μM and P<0.02 with 10 μM IM) as compared to CD34+ cells from IM-responders (n=8) in the presence of 5 and 10 μM IM, as determined by colony-forming cell (CFC) assays. Although more IM-resistant CFCs were obtained in the presence of IM from 3-week cultures initiated with CD34+ cells from the same IM-nonresponders than from IM responders, these latter differences were not significantly different (P= 0.28). These results suggest that the CD34+ leukemic cells from individual chronic phase CML patients harbor differences in their biologic properties that are predictive of how they will respond to IM therapy and that assessment of these differences may form the basis of rapid, practical and quantitative tests to assist in optimized patient management.

FGF2 Mediates Resistance In CML Patients In The Absence Of Kinase Domain Mutations, and Resistance Is Overcome By Ponatinib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3983-3983
Author(s):  
Elie Traer ◽  
Nathalie Javidi-Sharifi ◽  
Anupriya Agarwal ◽  
Jennifer B Dunlap ◽  
Isabel English ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Kinase Inhibitors ◽  
K562 Cells ◽  
Kinase Domain ◽  
Bone Marrow Microenvironment ◽  
Abl Kinase ◽  
Mechanism Of Resistance ◽  
Kinase Domain Mutations

Abstract Background Development of resistance to kinase inhibitors remains a challenge in chronic myeloid leukemia (CML). Kinase domain mutations are a common mechanism of resistance, yet the mechanism of resistance in the absence of mutations remains less clear. Recent evidence suggests that the bone marrow microenvironment provides a sanctuary for leukemia cells, and may be involved in mediating resistance to imatinib – particularly in the absence of BCR-ABL kinase domain mutations. We tested selected cytokines, growth factors, and extracellular matrix proteins expressed by cells in the bone marrow microenvironment for their ability to protect CML cells from imatinib. Results We found that fibroblast growth factor 2 (FGF2) was the most protective protein for the K562 CML cell line when exposed to imatinib. FGF2 was not only capable of promoting growth in short-term culture, but uniquely able to promote long-term resistance in vitro (p<0.0001 by 2-way ANOVA analysis). To analyze the mechanism of resistance, we used siRNA to target the FGF receptors 1-4 and found that only siRNA targeting FGFR3 was able to abrogate the protective effect of FGF2. Phospho-chip and Western blot analysis revealed that FGF2 binds FGFR3, which then signals the downstream kinases Ras, c-RAF, MEK1, and ERK1/2 to promote survival in the presence of imatinib. Inhibition of FGFR3 with the specific FGFR inhibitor PD173074 led to dephosphorylation of this signaling cascade, and restored sensitivity to imatinib of FGF2-mediated resistant K562 cells. Resistance could also be overcome with ponatinib, a multi-kinase inhibitor that targets both BCR-ABL and FGFR, whereas imatinib, nilotinib and dasatinib were all ineffective against FGF2-mediated resistant K562 cells. Although ponatinib was rationally designed to circumvent the BCR-ABL T315I gatekeeper mutation, it was also able to achieve major cytogenetic responses in 62% of patients without detectable kinase domain mutations in the recent PACE trial. We theorized that increased FGF2 may drive resistance in the subset of patients without kinase domain mutations who respond to ponatinib, similar to our in vitro findings. To evaluate this possibility, we identified patients without kinase domain mutations who were responsive to ponatinib and quantified bone marrow FGF2 by immunohistochemistry. In comparison to ponatinib-responsive patients with kinase domain mutations, patients without kinase domain mutations had increased FGF2 in their bone marrow (50.5% versus 36.6%, p=0.033). Moreover, FGF2 in the marrow decreased concurrently with response to ponatinib, further suggesting that FGF2-mediated resistance is interrupted by FGFR inhibition (-15.9% versus 0.8%, when compared to the change in FGF2 of patients with kinase domain mutations, p=0.012). Qualitatively, FGF2 was predominantly localized in supportive stromal cells (consistent with previous reports), supporting a paracrine mechanism of resistance. Furthermore, we also evaluated a single patient without kinase domain mutations who was resistant to ponatinib. In this patient’s marrow, there was no elevation in FGF2 or change in FGF2 with ponatinib treatment. Taken together, inhibition of FGFR appears to be critical for the clinical activity of ponatinib in patients without kinase domain mutations. Conclusions In summary, our data supports a model of resistance in which FGF2 production by the marrow stromal cells promotes resistance to multiple ABL kinase inhibitors without the need for mutation of the ABL kinase domain. Resistance occurs via FGF2 ligand-induced activation of the FGFR3/Ras/MAPK pathway, and can be overcome by concomitant inhibition of ABL and FGFR. In combination with recent clinical data with ponatinib, our data suggest that FGF2-mediated resistance is a major mechanism of resistance in CML patients without kinase domain mutations. These results illustrate the clinical importance of ligand-induced resistance to kinase inhibitors and support an approach of developing rational inhibitor combinations to circumvent resistance, particularly in other kinase-driven malignancies that routinely develop resistance to kinase inhibitors. Disclosures: Tyner: InCyte Corporation: Research Funding. Druker:Novartis, Bristol-Myers Squibb, & ARIAD: Novartis, BMS & ARIAD clin trial funding. OHSU holds contracts; no salary/lab research funds. OHSU & Druker have financial interest in MolecularMD; technology used in some studies licensed to MolecularMD. This conflict reviewed and managed by OHSU. Other.

Alternatively spliced truncated BCR-ABL1 protein in CML patients with resistance to kinase inhibitors

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 7026-7026
Author(s):  
J. Bruey ◽  
H. Kantarjian ◽  
W. Ma ◽  
C. Yeh ◽  
R. Peralta ◽  
...  
Keyword(s):  
Structural Changes ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Residual Disease ◽  
Chronic Phase ◽  
K562 Cells ◽  
Aurora Kinase ◽  
Imatinib Resistant ◽  
Alternatively Spliced

7026 Background: We have reported that some patients with imatinib-resistant chronic myeloid leukemia (CML) express an alternatively spliced BCR-ABL mRNA with a 35-bp insertion (BCR-ABL135INS), resulting in the addition of 10 residues and truncation of 653 residues. Molecular dynamic simulation suggested that this truncation and insertion of new 10 AA results in structural changes similar to those seen in BCR-ABL with T315I mutation. Here we evaluate the prevalence of BCR-ABL135INS in imatinib-resistant CML, examine the effect of this mutation on resistance to compared the efficiency of various kinase inhibitors in vitro, and suggest a model for persistent CML and a possible strategy to eradicate residual disease. Methods: Using a sensitive PCR method, we determined the prevalence of the alternatively spliced BCR-ABL135INS mRNA in 288 patients with chronic-phase CML resistant to imatinib. Expression of truncated protein was confirmed by Western blot. We then tested the effectiveness of various kinase inhibitors on human K562 CML cells expressing different levels of BCR-ABL135INS along with wild-type BCR-ABL1. Results: BCR-ABL135INS mRNA was detected in 210 (73%) of the 288 patients. Only 25% of BCR-ABL135INS positive cases showed coexistance of ABL1 kinase point mutation. Immunoprecipitation studies demonstrated that expression of the predicted 143-kD BCR-ABL135INS protein at levels proportional to those predicted by mRNA. Expression of BCR-ABL135INS in K562 cells was sufficient to conferred resistance to imatinib, dasatinib, and nilotinib in a dose-dependant fashion. However, no resistance was detected using aurora kinase inhibitor (MK 0457) or homoharringtonine (HHT). BCR-ABL135INS suppressed imatinib, nilotinib, and dasatinib-mediated dephosphorylation of CRKL, LYN, SRC, and STAT5, but had no effect on MK 0457-mediated dephosphorylation. The combination of imatinib with nilotinib or HHT showed strong synergy, overcoming BCR-ABL135INS-induced resistance in vitro. Conclusions: These findings emphasize the importance of the overlooked alternatively spliced BCR-ABL135INS protein and may provide a strategy to treat resistant disease and eradicate residual CML. No significant financial relationships to disclose.

scholarly journals Emodin Exerts an Antiapoptotic Effect on Human Chronic Myelocytic Leukemia K562 Cell Lines by Targeting the PTEN/PI3K-AKT Signaling Pathway and Deleting BCR-ABL

2016 ◽  
Vol 16 (4) ◽  
pp. 526-539 ◽  
Author(s):  
Chun-Guang Wang ◽  
Liang Zhong ◽  
Yong-Li Liu ◽  
Xue-Jun Shi ◽  
Long-Qin Shi ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
K562 Cells ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Myelocytic Leukemia ◽  
Novel Drugs ◽  
Alternative Drug ◽  
Drug Treatments

The BCR-ABL kinase inhibitor, imatinib mesylate, is the front-line treatment for chronic myeloid leukemia, but the emergence of imatinib resistance has led to the search for alternative drug treatments. There is a pressing need, therefore, to develop and test novel drugs. Natural products including plants, microorganisms, and halobios provide rich resources for discovery of anticancer drugs. In this article, we demonstrate that emodin inhibited the growth of K562 cells harboring BCR-ABL in vitro and in vivo, and induced abundant apoptosis, which was correlated with the inhibition of PETN/PI3K/Akt level and deletion of BCR-ABL. These findings suggest that emodin is a promising agent to kill K562 cells harboring BCR-ABL.

A Novel 4-anilino-3-quinolinecarbonitrile Dual Src and Abl Kinase Inhibitor (SKI-606) Has In Vitro Activity on CML Ph+Blast Cells Resistant to Imatinib.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1991-1991 ◽  
Author(s):  
Tiziana Grafone ◽  
Manuela Mancini ◽  
Emanuela Ottaviani ◽  
Matteo Renzulli ◽  
Frank Boschelli ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
G1 Phase ◽  
Inhibition Of Proliferation ◽  
Cd34 Cells ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

Abstract The tyrosine kinase Bcr-Abl is the fusion product of a reciprocal translocation between chromosomes 9 and 22, known as Philadelphia chromosome and it is present in the leukemic cells of more than 95% of patients with chronic myeloid leukemia (CML). Overexpression of Bcr-Abl in myeloid cells activates various signaling pathways. Previous studies have demonstrated that certain Src family kinases, such as Hck and Lyn, are also targets of Bcr-Abl activity. Hck and Lyn are expressed and activated in CML blast-crisis patients and their increased expression correlates with disease progression or STI571 resistance in some CML patients. Resistance to STI571 seems to be mediated by amplification of or mutations in the Bcr-Abl gene, reducing sensitivity to this inhibitor; newer Abl inhibitors may be susceptible to the same mechanism of resistance. Alternative strategies for control of CML, including the biological relevance of the Bcr-Abl - Src family kinase pathway, are necessary. One such strategy is the use of a specific small molecule Src kinase inhibitor. Recently, a new class of compounds, 4-anilino-3-quinolinecarbonitrile Src kinase inhibitors, has been synthesized. One member of this class, SKI606, is a dual-specificity inhibitor of both Src family and Abl kinases. To investigate the effect in vitro of SKI-606, we analyzed human cell lines from CML patients in blast crisis (K562, MK2, LAMA) and CD34+ from 9 patients in CML blast crisis using a wide range of concentrations (0.01μM-10μM) of this novel agent. Cell cycle analysis, in particular for the cell lines, showed that a major effect of SKI606 is to alter cell cycle progression, producing G1/S arrest. SKI606 induced dose-dependent inhibition of proliferation with IC50 of 1μM at 24hr. Flow cytometric analysis with Annexin-V showed that SKI-606 induced apoptosis of 50% of cells at 48hr. Western blotting and immuno-blotting analyses showed reduced phosphorylation of Bcr-Abl and also of Lyn and Hck. We also demonstrated activation of Caspase-9, an effector cysteine-protease, after exposure to SKI606. These drug effects also reduced the oncogenic effects of the Bcr-Abl gene product in CD34+ cells from patients with CML blast crisis. SKI606 induced a dose-dependent inhibition of proliferation with an IC50 of 1μM at 48hr and induction of apoptosis at 72hr. Cytofluorimetric analysis after 72hr of exposure revealed marked accumulation of cells in the G1 phase of cell cycle, accompanied by a significant increase in the number of apoptotic cells. In some of these patient samples, we observed hypophosphorylation of Bcr-Abl, Hck and Lyn at low concentration of SKI606 (1uM at 24h, 10uM at 48h). Interestingly, CD34+ cells taken from two of our imatinib-resistant patients with Bcr-Abl point mutations (E255K and Y253H) in the P-loop region of the protein exhibited a significant increase of apoptosis (50%) and a block in G1 phase of the cell cycle after treatment with 1 μM SKI606 for 48h. Our study thus showed a potential therapeutic usefulness of the drug in treatment of CML, particularly in blast crisis phase. Ongoing gene expression profiles will contribute to further understanding of the drug mechanism.

Identification and Functional Significance of Genes Regulated by Structurally Different ABL Kinase Inhibitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2860-2860
Author(s):  
Kousuke Nunoda ◽  
Tetsuzo Tauchi ◽  
Tomoiku Takaku ◽  
Masahiko Sumi ◽  
Seiichi Okabe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Inhibition ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Specific Inhibitor ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Altered Expression ◽  
Abl Kinase ◽  
Abl Kinase Inhibitors

Abstract Imatinib is an ABL-specific inhibitor that binds with high affinity to the inactive conformation of the BCR-ABL tyrosine kinase and has been shown to be effective in the treatment of chronic myelogenous leukemia. Dasatinib is an ATP-competitive, dual-spesific SRC and ABL kinase inhibitor that can bind BCR-ABL in both the active and inactive conformations. From a clinical stand point, dasatinib is particular attractive because it has been shown to induce hematologic and cytogenetic responses in imatinib-resistant CML patients. In the view of the fact that the combination of imatinib and dasatinib shows the additive/synergistic growth inhibition on a wild type p210 BCR-ABL expressing cells, we reasoned that these ABL kinase inhibitors might induce the different molecular pathways. To address this question, we used DNA microarrays to identify genes whose transcription was altered by imatinib and dasatinib. K562 cells were cultured with imatinib or dasatinib for 16 hrs, and gene expression data was obtained from three independent microarray hybridizations. Almost all of the imatinib- and dasatinib- responsive genes appeared to be similarly increased or decreased in K562 cells; however, small subsets of genes were identified as selectively altered expression by either imatinib or dasatinib. The genes whose expression was affected by imatinib and dasatinib were categorized into different functional groups based on their biological function, and genes in the cell proliferation and apoptosis categories were examined in detail. Imatinib and dasatinib affected the expression of several cyclin-dependent kinases (CDK2, CDK4, CDK6, CDK8, and CDK9), cell division cycle genes (CDC6, CDC7, CDC25C, and CDC34), and cyclones (cyclin A2, C, D2, D3, E1, E2, F, G1, G2, and H). Imatinib and dasatinib also modulated the expression of apoptosis-related genes including APAF1, BAK1, BCL2, BCL10, MCL1, CASP3, and CASP6). One of the distinct genes which are selectively modulated by dasatinib are CDK2 and CDK8, which had a maximal fold reduction of &lt;8-fold in microarray screen. Immunoblotting confirmed that gene expression changes induced only by dasatinib correlated with changes in protein expression. To assess the functional importance of dasatinib regulated genes, we used RNA interference to determine whether reduction of CDK2 and CDK8 affected the growth inhibition. The siRNA to CDK2 or CDK8 specifically reduced cdk2 or cdk8 in K562 cells. K562 cells pretreated with CDK2 or CDK8 siRNA showed the additive growth inhibition with imatinib but not with dasatinib. These finding demonstrate that the additive/synergistic growth inhibition by imatinib and dasatinib may be mediated by CDK2 and CDK8.

In Vitro and In Vivo Activity of the Src/Abl Kinase Inhibitor AP23464 and Analogs Against Activation Loop Mutants of KIT.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 793-793 ◽  
Author(s):  
Amie S. Corbin ◽  
Shadmehr Demehri ◽  
Ian J. Griswold ◽  
Chester A. Metcalf ◽  
William C. Shakespeare ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Myelogenous Leukemia ◽  
Activation Loop ◽  
Wild Type ◽  
Abl Kinase ◽  
Ic50 Values

Abstract Oncogenic mutations of the KIT receptor tyrosine kinase have been identified in several malignancies including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), seminomas/dysgerminomas and acute myelogenous leukemia (AML). Mutations in the regulatory juxtamembrane domain are common in GIST, while mutations in the activation loop of the kinase (most commonly D816V) occur predominantly in SM and at low frequency in AML. Several ATP-competitive kinase inhibitors, including imatinib, are effective against juxtamembrane KIT mutants, however, the D816V mutant is largely resistant to inhibition. We analyzed the sensitivities of cell lines expressing wild type KIT, juxtamembrane mutant KIT (V560G) and activation loop mutant KIT (D816V,F,Y and murine D814Y) to a potent Src/Abl kinase inhibitor, AP23464, and analogs. IC50 values for inhibition of cellular KIT phosphorylation by AP23464 were 5–11 nM for activation loop mutants, 70 nM for the juxtamembrane mutant and 85 nM for wild type KIT. Consistent with this, IC50 values in cell proliferation assays were 3–20 nM for activation loop mutants and 100 nM for wild type KIT and the juxtmembrane mutant. In activation loop mutant-expressing cell lines, AP23464, at concentrations ≤50 nM, induced apoptosis, arrested the cell cycle in G0/G1 and down-regulated phosphorylation of Akt and STAT3, signaling pathways critical for the transforming capacity of mutant KIT. In contrast, 500 nM AP23464 was required to induce equivalent effects in wild-type KIT and juxtamembrane mutant-expressing cell lines. These data demonstrate that activation loop KIT mutants are considerably more sensitive to inhibition by AP23464 than wild type or juxtamembrane mutant KIT. Non-specific toxicity in parental cells occurred only at concentrations above 2 μM. Additionally, at concentrations below 100 nM, AP23464 did not inhibit formation of granulocyte/macrophage and erythrocyte colonies from normal bone marrow, suggesting that therapeutic drug levels would not impact normal hematopoiesis. We also examined in vivo target inhibition in a mouse model. Mice were subcutaneously injected with D814Y-expressing (D816V homologous) murine mastocytoma cells. Once tumors were established, compound was administered three-times daily by oral gavage. One hour post treatment we observed >90% inhibition of KIT phosphorylation in tumor tissue. Following a three-day treatment regimen, there was a statistically significant difference in tumor size compared to controls. Thus, AP23464 analogs effectively target D816-mutant KIT both in vitro and in vivo and inhibit activation loop KIT mutants more potently than the wild type protein. These data provide evidence that this class of kinase inhibitors may have therapeutic potential for D816V-expressing malignancies such as SM or AML.

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