Overcoming Bcr-Abl T315I mutation by combination of GNF-2 and ATP competitors in an Abl-independent mechanism

Mamduh Khateb,Hazem Khamisie,Yousef Najajreh,Jamal Mahajna,Nili Ruimi,Afsar Mian,Martin Ruthardt,Anna Metodieva

doi:10.1186/1471-2407-12-563

Abstract

BackgroundPhiladelphia 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.MethodsBa/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.ResultsIn 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.ConclusionsOur 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.

Highlights

Philadelphia positive leukemias are characterized by the presence of Bcr-Abl fusion protein which exhibits an abnormal kinase activity
Our data illustrated the ability of allosteric inhibitors such as GNF-2 to cooperate with Abl kinase inhibitors (AKIs) to overcome T315I mutation by Bcr-Abl-independent mechanisms, providing a possibility of enhancing AKIs efficacy and overcoming resistance in Ph+ leukemia cells
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

Summary

Introduction

Philadelphia positive leukemias are characterized by the presence of Bcr-Abl fusion protein which exhibits an abnormal kinase activity. GNF-2, Abl allosteric kinase inhibitor, was demonstrated to possess cellular activity against Bcr-Abl transformed cells. Philadelphia positive leukemias are hematological malignancies caused by a chromosomal rearrangement that generates a fusion protein, Bcr–Abl, with deregulated tyrosine kinase activity. 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 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]. 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]

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Conclusion