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.
Khateb et al BMC Cancer 2012, 12:563 http://www.biomedcentral.com/1471-2407/12/563 RESEARCH ARTICLE Open Access Overcoming Bcr-Abl T315I mutation by combination of GNF-2 and ATP competitors in an Abl-independent mechanism Mamduh Khateb1, Nili Ruimi1, Hazem Khamisie1, Yousef Najajreh2, Afsar Mian3, Anna Metodieva3, Martin Ruthardt3 and Jamal Mahajna1,4* Abstract 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 Keywords: Philadelphia chromosome, Bcr-Abl, “gatekeeper” mutation T315I, Allosteric inhibition, Abl kinase inhibitors Background Philadelphia positive leukemias are hematological malignancies caused by a chromosomal rearrangement that generates a fusion protein, Bcr–Abl, with deregulated tyrosine kinase activity Imatinib, which targets the ATPbinding site, is effective in the early stage of the treatment of Ph-positive patients, but advanced-stage patients may relapse as a result of the emergence of point * Correspondence: jamalm@migal.org.il Cancer Drug Discovery Program, Galilee Technology Center, Migal, P.O.Box 831, Kiryat Shmona 11016, Israel Department of Nutritional Sciences, Tel-Hai College, Kiryat Shmona, Israel Full list of author information is available at the end of the article mutations within the Bcr–Abl Two recently approved drugs, Nilotinib [1] and Dasatinib [2] inhibit the activity of mutated Bcr-Abl that is refractory to Imatinib except the ‘gatekeeper’ T315I mutation, which is situated in the middle of the ATP-binding cleft [3] Allosteric kinase inhibitors hold promise for revealing unique features of kinases that may not be apparent using conventional ATP-competitive inhibitors Thus, using an unbiased cellular screening approach, GNF-2, a non-ATP-competitive inhibitor, has been identified and shown to demonstrate cellular activity against Bcr-Abl transformed cells [4] The exquisite selectivity of GNF-2 is due to the finding that it targets the myristate binding © 2012 Khateb et al.; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Khateb et al BMC Cancer 2012, 12:563 http://www.biomedcentral.com/1471-2407/12/563 site located near the C-terminus of the Abl kinase domain, as demonstrated by genetic approaches, solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry [5] GNF-2, like myristate, is able to induce and/or stabilize the clamped inactive conformation of Abl, analogous to the SH2-Y527 interaction of Src [6] Crystallography study revealed that GNF-2 replaces the myristoylated peptide in the crystals [5] As expected, most of the interactions between GNF2 and the protein are hydrophobic Mutations of three residues near the mouth of the myristate-binding site (C464Y, P465S and V506L) were reported to cause resistance to the binding of GNF-2, presumably for steric reasons The myristate-binding-site mutant, E505K, was inhibited by Imatinib and Nilotinib, but not by GNF-2, arguing that GNF-2 targets the myristoyl pocket [5] In this report we showed that GNF-2 cooperated with the Abl kinase inhibitors (AKIs), Imatinib, Nilotinib and Dasatinib, in inhibiting clonigenicity of Bcr-Abl T315I transformed Ba/F3 cells Interestingly, activity against T315I mutation was Bcr-Abl independent Furthermore, GNF-2 and AKIs also cooperated to inhibit JAK2 phosphorylation in Ba/F3 carrying T315I mutation Materials and methods Cell lines and cell cultures Ba/F3 cells expressing Bcr-Abl constructs or activated JAK2 (V617F) were previously described [7] and grown in RPMI 1640 with mM L-glutamine supplemented with 10% fetal bovine serum Penicillin at 100 U/ml, and streptomycin at 100 μg/ml, was added to the culture media SupB15, a Ph+ ALL B cell (ATCC, Rockville, MD) was grown in RPMI 1640 containing mM L-glutamine, 20% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin All cell lines were grown at 37°C in a humidified atmosphere with 5% CO2 Cellular Bcr-Abl auto-phosphorylation and immune-blotting Ba/F3 cells expressing the native or the T315I mutated Bcr-Abl protein (4 x 105 cells/ml) were treated with Abl kinase inhibitors (AKIs), GNF-2, combinations of GNF-2 and AKIs and DMSO for h Cells were collected, washed once with cold PBS, and lysed as previously describe [7] Cell lysate supernatants (40 μg protein) were resolved on 8% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and analyzed by immune-blotting with Anti-phospho-c-Abl (Tyr245), Anti-phospho-STAT5α (Tyr694) and antiphospho JAK2 (Tyr1007/1008) antibodies (Cell Signaling Technology, USA) The phosphorylated level of Bcr-Abl protein was compared to total Abl or α-tubulin that were detected using Anti-c-Abl and Anti-α-tubulin antibodies (Santa Cruz Biotechnology, USA) Quantitative analysis of Page of 10 the protein bands detected by Western blot was carried out using Tina software 2.0 Analyses of pSTATα pBcr-Abl and pJAK2 levels are given as folds of the sample values versus the α -tubulin values used as a loading control Trypan blue exclusion assay Ba/F3 cells containing Bcr-Abl constructs were plated (4x 105 cells/well) in six-well plates, with each well containing ml medium After 24 h, cells were treated with the appropriate agents Solvent-treated samples were incubated with 1% DMSO Seventy-two hours later, the cells were collected, stained with 0.4% trypan blue solution (1:1), and counted using a hemocytometer to determine IC50 values Colony-forming assay A colony-forming assay was performed as previously described [7] Briefly, cells (1 x 104) in ml RPMI/10% FBS medium were diluted in ml of 0.6% agar to give a final agar concentration of 0.3% agar The cell-agar mixture was poured on top of a hardened agar base in wells of 12-well plates and allowed to solidify Once the top layer solidified, ml of medium containing different treatments was placed on top to keep the agar moist The cells were grown at 37°C in a 5% CO2 humidified atmosphere until colonies were visible (2 weeks) The plates were stained for h with mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), and the dye was extracted with ml solubilization buffer (20% sodium dodecyl sulfate [SDS], 50% N,N-dimethyl-formamide, 25 mM HCL) for 24 h The optical density was measured at 570 nm wavelength with a reference wavelength of 630 nm Statistical analysis Statistical analysis was performed using Student’s t-test, with significant values set at *P