Rosalind H. Gunby

Molecular mechanisms of resistance to imatinib in Philadelphia-chromosome-positive leukaemias

Imatinib (STI571 or CGP57148B) is an innovative treatment for tumours with a constitutively activated form of c-ABL, c-KIT, or PDGFR. Such tumours include Philadelphia-chromosome-positive (Ph-positive) leukaemias, gastrointestinal stromal tumours, and PDGFR-positive leukaemias. Diseases such as primary hypereosinophilia and dermatofibrosarcoma protuberans also seem to respond to imatinib. Clinical trials assessing the therapeutic effects of imatinib have shown that the drug is highly effective with few associated side-effects, achieving durable cytogenetic responses in many patients with chronic-phase BCR-ABL-positive leukaemias. However, the emergence of resistance, particularly in patients with acute leukaemias, has prompted intense research, and many are concerned about the future prospects for imatinib. The resistance has been found in patients with acute-phase disease, but may also occur in patients with chronic-phase disease. Two cellular mechanisms for resistance to imatinib have been identified: amplification of BCR-ABL gene and mutations in the catalytic domain of the protein. In addition, suboptimum inhibition of BCR-ABL in vivo could contribute to the selection of resistant cells. We have summarised all currently available data on resistance to imatinib, both published and unpublished, including the mechanisms of resistance identified so far, and their clinical relevance to the different forms of Ph-positive leukaemias is discussed. Furthermore, we discuss strategies to overcome or prevent the development of resistance.

Characterization of Some Molecular Mechanisms Governing Autoactivation of the Catalytic Domain of the Anaplastic Lymphoma Kinase

NPM/ALK is an oncogenic fusion protein expressed in ∼50% of anaplastic large cell lymphoma cases. It derives from the t(2;5)(p23;q35) chromosomal translocation that fuses the catalytic domain of the tyrosine kinase, anaplastic lymphoma kinase (ALK), with the dimerization domain of the ubiquitously expressed nucleophosmin (NPM) protein. Dimerization of the ALK kinase domain leads to its autophosphorylation and constitutive activation. Activated NPM/ALK stimulates downstream survival and proliferation signaling pathways leading to malignant transformation. Herein, we investigated the molecular mechanisms of autoactivation of the catalytic domain of ALK. Because kinases are typically regulated by autophosphorylation of their activation loops, we systematically mutated (Tyr → Phe) three potential autophosphorylation sites contained in the “YXXXYY” motif of the ALK activation loop, and determined the effect of these mutations on the catalytic activity and biological function of NPM/ALK. We observed that mutation of both the second and third tyrosine residues (YFF mutant) did not affect the kinase activity or transforming ability of NPM/ALK. In contrast, mutation of the first and second (FFY), first and third (FYF), or all three (FFF) tyrosine residues impaired both kinase activity and transforming ability of NPM/ALK. Furthermore, a DFF mutant, in which the aspartic residue introduces a negative charge similar to a phosphorylated tyrosine, possessed catalytic activity similar to the YFF mutant. Together, our findings indicate that phosphorylation of the first tyrosine of the YXXXYY motif is necessary for the autoactivation of the ALK kinase domain and the transforming activity of NPM/ALK.

Anaplastic lymphoma kinase and its signalling molecules as novel targets in lymphoma therapy

A crucial issue in the development of molecularly-targeted anticancer therapies is the identification of appropriate molecules whose targeting would result in tumour regression with a minimal level of systemic toxicity. Anaplastic lymphoma kinase (ALK) is a transmembrane receptor tyrosine kinase, normally expressed at low levels in the nervous system. As a consequence of chromosomal translocations involving the alk gene (2p23), ALK is also aberrantly expressed and constitutively activated in ~ 60% of CD30+ anaplastic large cell lymphomas (ALCLs). Due to the selective overexpression of ALK in tumour cells, its direct involvement in the process of malignant transformation and its frequent expression in ALCL patients, the authors recognise ALK as a suitable candidate for the development of molecularly targeted strategies for the therapeutic treatment of ALK-positive lymphomas. Strategies targeting ALK directly or indirectly via the inhibition of the protein networks responsible for ALK oncogenic signalling are dis-cussed.

Oncogenic Fusion Tyrosine Kinases as Molecular Targets for Anti-Cancer Therapy

Deregulated activation of protein tyrosine kinases (PTKs) is a frequent event underlying malignant transformation in many types of cancer. The formation of oncogenic fusion tyrosine kinases (FTKs) resulting from genomic rearrangements, represents a common mechanism by which kinases escape the strict controls that normally regulate their expression and activation. FTKs are typically composed of an N-terminal dimerisation domain, provided by the fusion partner protein, fused to the kinase domain of receptor or non-receptor tyrosine kinases (non-RTKs). Since FTKs do not contain extracellular domains, they share many characteristics with non-RTKs in terms of their properties and approaches for therapeutic targeting. FTKs are cytoplasmic or sometimes nuclear proteins, depending on the normal distribution of their fusion partner. FTKs no longer respond to ligand and are instead constitutively activated by dimerisation induced by the fusion partner. Unlike RTKs, FTKs cannot be targeted by therapeutic antibodies, instead they require agents that can cross the cell membrane as with non-RTKs. Here we review the PTKs known to be expressed as FTKs in cancer and the strategies for molecularly targeting these FTKs in anti-cancer therapy.

Characterization of compound 584, an Abl kinase inhibitor with lasting effects

Secondary resistance to imatinib is observed in patients with chronic myeloid leukemia. This study describes a novel Abl kinase inhibitor with long-lasting effects. Background Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosome-positive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584). Design and Methods To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotrans-planted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor. Results cmp-584 showed potent anti-Abl activity both on recombinant protein (IC50: 8 nM) and in cell-based assays (IC50: 0.1–10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC50: 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib. Conclusions The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former.