Applying the discovery of the Philadelphia chromosome

Abstract

The identification of the Philadelphia chromosome in cells from individuals with chronic myelogenous leukemia (CML) led to the recognition that the BCR-ABL tyrosine kinase causes CML. This in turn led to the development of imatinib mesylate, a clinically successful inhibitor of the BCR-ABL kinase. Incorporating the use of markers of BCR-ABL kinase inhibition into clinical trials led to the realization that imatinib-resistant kinase domain mutations are the major cause of relapse during imatinib therapy and the subsequent development of new inhibitors to treat CML patients. The development of imatinib validates an emerging paradigm in cancer, in which a tumor is defined by genetic abnormalities and effective therapies are developed that target events critical to the growth and survival of a specific tumor.

Figure 1. The phenotype and genotype of CML.

(A) A bone marrow biopsy from a patient with CML shows the typical hypercellularity with granulocytic and megakaryocytic hyperplasia (original magnification, ×200). (B) The peripheral blood is characterized by a full spectrum of myeloid cells, including immature myeloid cells with rare blasts. Basophilia is also observed (original magnification, ×630). (C) Dual-color, dual-fusion FISH displaying BCR-ABL signals in bone marrow cells in metaphase (left) and interphase (right). The red fluorescent probe is specific for ABL, while the green probe is specific for BCR. Yellow signals the presence of BCR-ABL and ABL-BCR fusions.

Figure 2. A model of the domains of the c-ABL and BCR-ABL proteins displays the functional consequences of the translocation.

(A) In c-ABL, the P-loop and the activation loop surround the active site for substrate phosphorylation (shaded). Under normal conditions c-ABL is inactive, with the SH2 and SH3 domains bound to the kinase domain, restricting its kinase activity (53). This conformation is stabilized by the cap domain, which is anchored to the kinase domain by a myristoyl group (jagged line) (52, 53). (B) In BCR-ABL, the myristoyl group of the cap domain is lost and is replaced by BCR, shown for simplicity as a single globular domain. This is presumed to destabilize binding of SH2 and SH3 to the kinase domain (56). The N terminus of BCR, the coiled-coil domain, forms a helical dimer (that also tetramerizes) and tethers individual BCR-ABL proteins together (54). This allows for transphosphorylation (P; yellow circles) of the kinase, which promotes the kinase to adopt an active conformation with a significant outward twist of the activation loop. (C) Imatinib binds to the active site of the kinase domain, freezing it in the inactive conformation, preventing BCR-ABL activation (25).

Figure 3. Magnified views into the active sites of the ABL kinase domain in complex with imatinib (A) and dasatinib (B).

The two compounds have very different modes of binding to the kinase, with dasatinib more confined to the ATP-binding pocket than imatinib. In addition, imatinib binds the inactive and dasatinib the active conformations, with opposite orientations of the catalytic Glu-Phe-Gly amino acid residues (shown in light blue stick format). In the dasatinib structure, the glutamic acid of the Glu-Phe-Gly motif that coordinates a Mg²⁺ ion during catalysis is oriented properly for catalysis, whereas in the imatinib structure this residue points away from the active site. Both inhibitors reside in close proximity to the T315 residue. The side chain atoms of the residues susceptible to resistant mutations for each inhibitor are shown in green and inhibitor atoms shown in yellow stick format (nitrogen, dark blue; oxygen, red; sulfur, orange; chlorine, hot pink). The P-loop (residues 244–255) is shown in magenta and the activation loop (residues 381–402) is shown in light blue. (A) PDB entry 1IEP (24). (B) PDB entry 2GQG (78). The figure was created using PyMol (http://pymol.sourceforge.net/).