Chronic Myeloid Leukemia • Progress in Hematology New tyrosine kinase inhibitors in chronic myeloid leukemia Giovanni Martinelli The deregulated activity of BCR-ABL tyrosine kinase originating from the t(9;22) chro- Simona Soverini mosomal translocation has been shown to be necessary and sufficient for the trans- Gianantonio Rosti formed phenotype of chronic myeloid leukemia (CML) cells. This peculiarity has paved Daniela Cilloni the way for the development of novel therapies specifically targeting the BCR-ABL gene Michele Baccarani product. The first BCR-ABL inhibitor to come into use in clinical practice, imatinib mesy- late, is now the first-choice treatment for all newly diagnosed CML patients, but the ini- tial striking efficacy of this drug has been overshadowed by the development of clini- cal resistance. The most common mechanisms of resistance include (i) BCR-ABL over- expression, and (ii) BCR-ABL kinase domain mutations disrupting critical contact points between imatinib and BCR-ABL or inducing a transition to a conformation to which ima- tinib is unable to bind. Several approaches to overcoming resistance have been stud- ied both in vitro and in vivo. They include dose escalation of imatinib, the combination of imatinib with chemotherapeutic drugs, alternative BCR-ABL inhibitors, and inhibitors of kinases acting downstream of BCR-ABL such as Src kinases. Various novel tyrosine kinase inhibitors (TKI) have been synthesized and have now reached the pre-clinical or clinical phase. This review highlights the development of new TKI as specific molecu- larly targeted therapy and as the principal mechanisms for overcoming imatinib resist- ance. Key words: tyrosine kinase, CML. Haematologica 2005; 90:534-541 ©2005 Ferrata Storti Foundation From the Institute of Hematology hronic myeloid leukemia (CML) is a of the signal transduction pathways affect- and Medical Oncology “Seràgnoli”, hematopoietic disorder character- ed by the deregulated kinase activity of University of Bologna, Bologna, Italy ized by the malignant expansion of BCR-ABL has provided information on (GM, SS, GR, MB); Division of C Hematology, San Luigi Gonzaga bone marrow stem cells. CML is almost additional or alternative signaling steps that Hospital, Turin, Italy (DC). unique among human cancers since a single could be interrupted in an attempt to block genetic defect is responsible for the trans- the leukemogenic process. BCR-ABL exerts formed phenotype. The cytogenetic hall- its oncogenic effects in CML cells essential- Correspondence: mark of more than 90% of CML cases is a ly by stimulating cell proliferation, inhibit- Giovanni Martinelli, MD, Institute of Hematology and Medical reciprocal t(9;22)(q34;q11) chromosomal ing apoptosis and altering cell adhesion to Oncology “Seràgnoli”, University of translocation1 that creates a derivative 9q+ bone marrow stroma. The signal transduc- Bologna, Via Massarenti 9, and a small 22q-, most commonly referred tion cascades involved in these cellular 40138 Bologna, Italy. E-mail: to as the Philadelphia (Ph) chromosome.2 As processes and activated by BCR-ABL [email protected] a result of this translocation, the latter har- include, among others a) Ras;5 b) mitogen- bors a BCR-ABL fusion gene encoding a activated protein kinase (MAPK)6 and its chimeric BCR-ABL protein with deregulat- downstream effectors MEK and Erk; c) ed tyrosine kinase activity, the expression phosphatidylinositol-3 kinase (PI3K)7,8 and of which has been shown to be necessary its downstream effector Akt. and sufficient for the transformed pheno- With the sole exception of Ras, all these type of CML cells.3,4 Thanks to the contribu- proteins share an intriguing feature with tions of numerous researchers, the past BCR-ABL, i.e. they all are tyrosine kinases. twenty years have witnessed considerable Specific tyrosine kinase inhibitors (TKI) advances in our knowledge of the molecu- are drugs used to inhibit malignant cell lar and cell biology of CML, creating the growth and metastasis formation and are essential platform for engineering targeted currently undergoing a rapid phase of devel- molecular therapies. It soon became clear opment. Imatinib mesylate (formerly that the BCR-ABL oncoprotein itself is the STI571; GleevecTM or Glivec®, Novartis ideal target, since it plays a central role in Pharmaceuticals, Basel, Switzerland) is the CML pathogenesis, and it is not expressed first successful example of a TKI for the by normal cells. Furthermore, the dissection therapy of CML. It is a small-molecule | 534 | haematologica/the hematology journal | 2005; 90(4) New TKI in CML patients A B a.a. involved a.a. which a.a. mapping a.a. controlling in phosphate directly contact close to the kinase Phosphate binding loop binding imatinib catalytic domain activation/inactivation T315 E255 F317 G250 V289 p-loop catalytic activation Y253 domain loop Q252 Imatinib M351 Activation loop F486 M224V V289A H396 L248V M343T H396R/P E255K/V G250E/R M351T L387M Y253F/H T315I E355G/D Q252R/H F311L/I F317L F382L V379I Catalytic loop Figure 1. A. Distribution of reported point mutations within the ABL kinase domain. Codons most frequently reported as being affected by amino acid (a.a.) substitutions are shown. Mutations cluster in four main regions. One group of mutations (G250E, Q252R, Y253F/H, E255K/V) includes amino acids that form the phosphate-binding loop for ATP (also known as the P-loop). A second group (V289A, F311L, T315I, F317L) can be found in the imatinib binding site; these mutations interact directly with the inhibitor via hydrogen bonds or Van der Waals’ interactions. The third group of mutations (M351T, E355G) clusters within the catalytic domain. The fourth group of mutations (H396R/P) is located in the activation loop, whose conformation is the molecular switch controlling kinase activation/inactivation. B. Three- dimensional representation of ABL complexed with imatinib. Position of mutations is highlighted, along with the activation loop, P-loop and catalytic loop. β-strands are numbered and α-helices are lettered according to the nomenclature used for insulin-receptor tyrosine kinase (adapted from Gambacorti-Passerini et al.).31 inhibitor of the BCR-ABL tyrosine kinase, as well as of mechanisms: (i) the overexpression of BCR-ABL, main- a limited number of other kinases (c-Kit, PDGF-R, ly due to gene amplification,19,20 and, more frequently, ARG).9,10 Preclinical and clinical studies11-18 have con- (ii) the acquisition or the selection of specific point firmed the remarkable efficacy and high tolerability of mutations within several critical regions of the ABL this drug. Imatinib mesylate is now the first-choice kinase domain (Figures 1A,B).19-27 treatment for all newly diagnosed CML patients, but Crystallographic studies have shown that the high the initial striking efficacy of this drug has been over- selectivity and efficacy of imatinib are due to its abili- shadowed by the development of clinical resistance. ty to bind and lock BCR-ABL in its inactive, auto-inhib- The emergence of resistance to imatinib has prompted ited conformation.28-29 Mutations seem to act by dis- researchers to focus on strategies aimed at preventing rupting critical contact points between imatinib and or overcoming this phenomenon. Since resistance often BCR-ABL or, more often, by inducing a transition from coincides with the reactivation of kinase activity with- the inactive to the active state, i.e. to a conformation to in the leukemic clone, either BCR-ABL itself or BCR- which imatinib is unable to bind. From analyses of ABL-triggered downstream signaling pathways contin- clinical samples, the repertoire of mutations found in ue to be good targets for molecular therapy. Several association with the resistant phenotype has been approaches have been studied both in vitro and in vivo. increasing slowly but inexorably over time (Table 1). They include dose escalation of imatinib, the combina- Mutations seem to cluster in four main regions. One tion of imatinib with chemotherapeutic drugs, alterna- group of mutations (G250E, Q252R, Y253F/H, tive BCR-ABL inhibitors including Src kinase inhibitors, E255K/V) includes amino acids that form the phos- and inhibitors of kinases downstream of BCR-ABL. phate-binding loop for ATP (also known as the P-loop). Further investigations into the molecular mechanisms A second group (V289A, F311L, T315I, F317L) can be of disease and how to specifically target the abnormal found in the imatinib binding site and interacts direct- cellular processes will serve as guides in the design of ly with the inhibitor via hydrogen bonds or Van der new treatment modalities in future clinical trials. Waals’ interactions. The third group of mutations (M351T, E355G) clusters in close proximity to the cat- BCR-ABL gene amplification and mutation are the pri- alytic domain. The fourth group of mutations mary mechanisms of imatinib resistance (H396R/P) is located in the activation loop, whose con- Despite high rates of hematologic and cytogenetic formation is the molecular switch controlling kinase responses, primary refractoriness and acquired resist- activation/inactivation. Conflicting data are still report- ance to imatinib are observed in a growing number of ed regarding the relative as well as the overall frequen- patients, especially those at more advanced stages of cy of resistance-associated mutations. It is also current- the disease. Resistance has been traced to two main ly unclear whether ABL mutations, or a specific subset haematologica/the