Introduction

In 1960, a minute acrocentric chromosome was noted in cells from seven patients with chronic myeloid leukemia (CML) (1). The subsequent identification of this abnormal chromosome 22, which came to be referred to as the Philadelphia chromosome, has become the basis for an explosion in knowledge over the past 40-plus years that culminated in the development of imatinib mesylate (IM), a highly effective targeted therapy of CML that is producing long-term disease control and a possible cure (2).

In 1973, it was recognized that the Philadelphia chromosome was actually the result of a reciprocal translocation between chromosomes 9 and 22 that has come to be designated as t(9;22) (q34;q11) (3). This reciprocal translocation resulted in the juxtaposition of the Abelson (ABL1) oncogene, a tyrosine kinase on chromosome 9, with a gene of unknown function on the long arm of chromosome 22, referred to as the breakpoint cluster region (BCR) (4). The identification of this hybrid abnormal gene, BCR-ABL1, was of major significance for multiple reasons.

Identification of this gene rearrangement is the sine qua non for the diagnosis of CML, and its presence in the hematopoietic cells of virtually all patients with CML made it an ideal target for the development of therapeutic agents. Proof that BCR-ABL1 was crucial for the development of CML was shown when the hybrid gene was transfected into bone marrow cells of mice which were subsequently transplanted into an irradiated syngeneic recipient and shown to lead to the development of a CML-like syndrome (5).

The breakpoint in the ABL1 gene that results in the BCR-ABL1 translocation is remarkably consistent from patient to patient and usually occurs in exon 2. In contrast, the breakpoint on the BCR gene can occur in multiple regions, and it is this variation that results in different-sized hybrid aberrant tyrosine kinase genes. The two most common breakpoints in BCR occur either in the "major" breakpoint cluster region (M-BCR) of the BCR gene between exons 12 and 16 (also known as b1 to b5) or in a "minor" BCR (m-BCR) region that exclude exons e1 and e2 of the BCR gene.

Two chimeric proteins result from these breakpoints including a 210-kd gene known as the p210 BCR-ABL1 resulting from the break in the M-BCR and a smaller 190-kd BCR-ABL1 known as p190 BCR-ABL. The p210 BCR-ABL occurs most frequently in cases of CML whereas the 190-kd BCR-ABL is more commonly seen in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph±ALL) (6). Other less common breakpoints have also been seen in the BCR gene resulting in other chimeric proteins with the most common being a break in the e19-e20 exons (known as |x-BCR) and producing a 230-kd protein known as p230 BCR-ABL. This disorder most often results in a chronic neutrophilic leukemia (6) (Fig. 1A,B).

A primitive hematopoietic progenitor cell is thought to be the cell from which CML arises. Whether the BCR-ABL1 mutation is the initial mutation that occurs or is a subsequent mutation is unclear, but these abnormal cells are able to gain a growth advantage over normal hematopoietic cells with resultant suppression of normal hematopoiesis (7). Multiple mechanism are responsible for the dominance of the CML cells in hematopoiesis in afflicted patients and are not only likely a result of a proliferative advantage, but also lilely result in prolonged survival as a consequence of decreased apoptosis and altered adherence to marrow stromal elements. These latter mechanisms enhance the release of CML progenitors into the peripheral blood (8,9).

Fig. 1. The translocation of t(9;22)(q34;q11) in CML. The Philadelphia (Ph) chromosome is a shortened chromosome 22 that results from the translocation of 3' (toward the telomere) ABL segments on chromosome 9 to 5' BCR segments on chromosome 22. Breakpoints (arrowheads) on the ABL gene are located 5' (toward the centromere) of exon a2 in most cases. Various breakpoint locations have been identified along the BCR gene on chromosome 22. Depending on which breakpoints are involved, different-sized segments from BCR are fused with the 3' sequences of the ABL gene. This results in fusion messenger RNA molecules (e1a2, b2a2, b3a2, and e19a2) of different lengths that are transcribed into chimeric protein products (p190, p210, and p230) with variable molecular weights and presumably variable function. The abbreviation m-bcr denotes minor breakpoint cluster region, M-bcr major breakpoint cluster region, and ^-bcr a third breakpoint location in the BCR gene that is downstream from the M-bcr region between exons e19 and e20. (Reprinted with permission from Ref. (6)).

Fig. 1. The translocation of t(9;22)(q34;q11) in CML. The Philadelphia (Ph) chromosome is a shortened chromosome 22 that results from the translocation of 3' (toward the telomere) ABL segments on chromosome 9 to 5' BCR segments on chromosome 22. Breakpoints (arrowheads) on the ABL gene are located 5' (toward the centromere) of exon a2 in most cases. Various breakpoint locations have been identified along the BCR gene on chromosome 22. Depending on which breakpoints are involved, different-sized segments from BCR are fused with the 3' sequences of the ABL gene. This results in fusion messenger RNA molecules (e1a2, b2a2, b3a2, and e19a2) of different lengths that are transcribed into chimeric protein products (p190, p210, and p230) with variable molecular weights and presumably variable function. The abbreviation m-bcr denotes minor breakpoint cluster region, M-bcr major breakpoint cluster region, and ^-bcr a third breakpoint location in the BCR gene that is downstream from the M-bcr region between exons e19 and e20. (Reprinted with permission from Ref. (6)).

In normal cells, the ABL1 protein is found in both the nucleus and cytoplasm and can shuttle between these two locations (10). In contrast, the BCR-ABL1 protein is exclusively found in the cytoplasm where it is constitutively activated.

The ABL1 protein has three SRC-homology domains: SH1, SH2, and SH3 (Fig. 1C). The SH1 domain is a kinase domain and is organized into an N-lobe and C-lobe with the adenosine triphosphate (ATP)-binding catalytic site positioned between these two lobes. In the BCR portion of the molecule, the coil-motif encoded by the first BCR exon is responsible for dimerization of the BCR-ABL1 (11).

The normal c-ABL protein is structurally regulated by auto-inhibition in a fashion similar to that of c-SRC. This auto-inhibition depends on intramolecular interactions between the SH3 and SH2 domains and a linker between SH3 and SH2 known as the catalytic domain linker (CD-linker) and the myristoyl group from the cap or N-terminus region that is upstream of the SH3 domain (12).

A "switch-clamp-latch mechanism" is critical for the quiescent state of c-ABL normally. The switch refers to a flip that occurs in an activation loop from the C-lobe that goes between opened and closed conformations and represent active and inactive states of the enzyme, respectively (Fig. 2). This "clamp" or loop limits the access of ATP and other substrates to the active binding site of the ABL1 protein. A "latch" stabilizes

Mutations in:

9 ATP-Binding (P) loop Activation loop

• Hinge region 244 Resjdue numbef Qf

• Activation loop mutated c-Abl kinase

Mutations in:

9 ATP-Binding (P) loop Activation loop

• Hinge region 244 Resjdue numbef Qf

• Activation loop mutated c-Abl kinase

Fig. 2. Structure of the ABL1 kinase portion of the BCR-ABL1 protein. The activation loop (blue) is in the closed (inactive) conformation on the left and in the open (active) conformation on the right. A molecule of imatinib is positioned in the ATP-binding site and is in green. (Reprinted with permission from U.S. Healthcare Communications, LLC. LitzowMR, Tefferi A. Chronic myeloid leukemia: problems propel progress. Amer JHematol/Oncol, 2007; 6(5) supplement 7:19-22).

Fig. 3. A simplified illustration of BCR-ABL and SRC family kinase involvement in oncogenic signaling pathways. The inhibitory effect is indicated by the upside-down T's. ABL = Abelson tyrosine kinase; BCR = breakpoint cluster region; FAK = focal adhesion kinase; Grb-2 = growth factor receptor-bound protein 2; HcK = hematopoietic cell kinase; JNK = Jun amino-terminal kinase; P = phosphate group; PI3 K = phosphatidylinositol-3-kinase; SFK = SRC family kinases; Stat5 = signal transducer and activator of transcription 5. (Reprinted with permission from Ref. (123)).

Fig. 3. A simplified illustration of BCR-ABL and SRC family kinase involvement in oncogenic signaling pathways. The inhibitory effect is indicated by the upside-down T's. ABL = Abelson tyrosine kinase; BCR = breakpoint cluster region; FAK = focal adhesion kinase; Grb-2 = growth factor receptor-bound protein 2; HcK = hematopoietic cell kinase; JNK = Jun amino-terminal kinase; P = phosphate group; PI3 K = phosphatidylinositol-3-kinase; SFK = SRC family kinases; Stat5 = signal transducer and activator of transcription 5. (Reprinted with permission from Ref. (123)).

the interaction with a myristoyl chain from the cap region. If any of these modules are disrupted, tyrosine kinase activity of the ABL is activated leading to cell growth (13). Thus, the BCR-ABL1 protein is able to phosphorylate tyrosine on a large number of substrate molecules and enhance the proliferative potential of the malignant cell (Fig. 3).

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