The twenty-first century is beginning with a sharp turn in the field of cancer therapy. Molecular targeted therapies against specific oncogenic events are now possible. The BCR-ABL story represents a notable example of how research from the fields of cytogenetics, retroviral oncology, protein phosphorylation, and small molecule chemical inhibitors can lead to the development of a successful molecular targeted therapy. Imatinib mesylate (Gleevec, STI571, or CP57148B) is a direct inhibitor of ABL (ABL1), ARG (ABL2), KIT, and PDGFR tyrosine kinases. This drug has had a major impact on the treatment of chronic myelogenous leukemia (CML) as well as other blood neoplasias and solid tumors with etiologies based on activation of these tyrosine kinases. Analysis of CML patients resistant to BCR-ABL suppression by Imatinib mesylate coupled with the crystallographic structure of ABL complexed to this inhibitor have shown how structural mutations in ABL can circumvent an otherwise potent anticancer drug. The successes and limitations of Imatinib mesylate hold general lessons for the development of alternative molecular targeted therapies in oncology.

Novartis Pharmaceuticals Corporation.

BCR-ABL is a fusion oncogene expressed in human leukemia cells with a (9;22) translocation known as the Philadelphia (Ph) chromosome. 2,3 Depending on the specific breakpoint, Bcr-Abl proteins exist in p190, p210, or p230 forms that are associated with different clinical syndromes. p210-Bcr-Abl is found in nearly all cases of chronic myelogenous leukemia (CML) and some patients with Ph ϩ acute lymphoid leukemia (ALL). p190-Bcr-Abl is almost exclusively found in ALL. Retroviral transduction of murine bone marrow cells with p210 or p190 forms can yield either myeloid or B-lymphoid tumors in recipient mice, depending on the experimental model used. 4 The Abl tyrosine kinase inhibitor imatinib (Gleevec, STI-571) has been shown to elicit remarkable clinical responses with minimal toxicity in CML and Ph ϩ ALL patients. Unfortunately, resistance eventually develops and is usually associated with mutations or amplifications of BCR-ABL. 5,6 Targeting multiple signaling steps therefore may generate more lasting remissions in patients with ABL-dependent leukemias. Expression of v-Abl or Bcr-Abl activates many of the same signaling intermediates including the small G proteins Ras and Rac, tyrosine kinases of the Janus kinase family, protein kinase C, and phosphoinositide 3-kinase (PI3K). 7,8 It is important to note that signaling components downstream of ABL oncogenes have been defined in diverse cell systems. However, it is becoming evident that many of the effects of v-ABL and BCR-ABL are cell-context specific and may not have functional significance in the transformation of primary cells. For example, signal transducer and activator of transcription 5 was shown to be dispensable for v-ABL-or BCR-ABL-mediated transformation and leukemogenesis even though it is phosphorylated in human CML cells and is required for conversion of hematopoietic cell lines to growth factor-independence. 9,10 Thus, it is critical that the contribution of specific signaling components to ABL-mediated disease be confirmed in direct transformation assays done in hematopoietic cell types representative of their natural target cells.The PI3K family of enzymes phosphorylates inositol phospholipids, thereby promoting membrane association of certain cytoplasmic proteins that specifically bind to PI3K lipid products. 11,12 There are many subtypes of PI3K with distinct functions in cells. Class I A PI3Ks, the subgroup that functions downstream of activated tyrosine kinases, are heterodimers composed of a catalytic subunit and a regulatory subunit. Signaling through class I A PI3K promotes cell proliferation and survival in many cell types and is strongly associated with transformation and metastasis. 13 There are 3 catalytic subunit isoforms (p110␣, p110␤, and p110␦) and 5 regulatory subunit isoforms (p85␣, p55␣, p50␣, p85␤, and p55␥; the first 3 are transcriptional variants of a single gene, Pik3r1). 11 We and others have shown that the predominant class I A regulatory isoform expressed in murine B cells, p85␣, is required for B-cell proliferation trigg...

RIN1 was originally identified by its ability to physically bind to and interfere with activated Ras in yeast. Paradoxically, RIN1 potentiates the oncogenic activity of the BCR-ABL tyrosine kinase in hematopoietic cells and dramatically accelerates BCR-ABL-induced leukemias in mice. RIN1 rescues BCR-ABL mutants for transformation in a manner distinguishable from the cell cycle regulators c-Myc and cyclin D1 and the Ras connector Shc. These biological effects require tyrosine phosphorylation of RIN1 and binding of RIN1 to the Abl-SH2 and SH3 domains. RIN1 is tyrosine phosphorylated and is associated with BCR-ABL in human and murine leukemic cells. RIN1 exemplifies a new class of effector molecules dependent on the concerted action of the SH3, SH2, and catalytic domains of a cytoplasmic tyrosine kinase.

Protein kinase inhibitors can be effective in treating selected cancers, but most suppress several kinases. Imatinib mesylate has been useful in the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia and B cell acute lymphoblastic leukemia through the inhibition of BCR-ABL tyrosine kinase activity. Imatinib mesylate has also been shown to inhibit KIT, ARG, and platelet-derived growth factor receptors ␣ and ␤, and potentially other tyrosine kinases. We have produced a mutant allele of BCR-ABL (T315A) that is uniquely inhibitable by the small molecule 4-amino-1-tert-butyl-3-(1-naphthyl)pyrazolo[3,4-D]pyrimidine and used it to demonstrate that sole suppression of BCR-ABL activity was insufficient to eliminate BCR-ABL ؉ KIT ؉ -expressing immature murine myeloid leukemic cells. In contrast, imatinib mesylate effectively eliminated BCR-ABL ؉ KIT ؉ -expressing leukemic cells. In the cellular context of mature myeloid cells and Pro͞Pre B cells that do not express KIT, monospecific BCR-ABL inhibition was quantitatively as effective as imatinib mesylate in suppressing cell growth and inducing apoptosis. These results suggest that the therapeutic effectiveness of small molecule drugs such as imatinib mesylate could be due to the inhibitor's ability to suppress protein kinases in addition to the dominant target.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR1–MR4), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR4.5 level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR4.5 sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

IntroductionCancer develops from an accumulation of genetic aberrations. 1 Particular genetic lesions may be essential for initiation and maintenance of particular cancers. Inducible transgenic models demonstrate that sustained activation of C-MYC is essential both for the establishment and continual growth of T-cell lymphomas, myeloid leukemias, and osteogenic sarcomas. 2,3 In human cancer cells at the time of diagnosis, the presence of multiple genetic defects usually prevents the identification of signaling pathways and cellular changes directly altered by the primary genetic lesion.In more than 90% of chronic myelogenous leukemia (CML) cases, the initial genetic aberration is the Philadelphia chromosome (Ph ϩ ), which results from a reciprocal translocation between chromosome 9 within the tyrosine kinase gene ABL and chromosome 22 within the BCR gene. 4,5 This creates a fusion gene product BCR-ABL. [6][7][8][9][10][11] The deregulated tyrosine kinase activity of BCR-ABL is required to transform cells in vitro 12 and to generate a CML-like disease in vivo. 13,14 Inhibition of BCR-ABL's tyrosine kinase activity with the drug imatinib mesylate (STI571; Novartis, Basel, Switzerland) is sufficient to suppress CML in patients [15][16][17] and relapses from therapy are due to reactivation of BCR-ABL via mutation or amplification of the gene. 18,19 These results demonstrate that the BCR-ABL oncogene is required both for establishment and maintenance of CML.CML begins with a long 3-to 5-year chronic phase characterized by an expansion of progenitor and mature myeloid cells, mild anemia, and splenomegaly. This chronic phase transforms to an aggressive stage (blast crisis) associated with numerous genetic abnormalities such as trisomy 8, trisomy 19, i(17q), and loss or mutation of p53 as well as epigenetic effects leading to an expansion of immature progenitors. 20,21 The cellular origin of CML begins with formation of the Ph ϩ in the hematopoietic stem cell (HSC) from where it is subsequently transmitted to all hematopoietic lineages. 22,23 Studies of purified Ph ϩ HSCs suggest that either BCR-ABL is not expressed in this population 24 or it is insufficient to affect the quiescent status of HSCs. 25 Multipotent hematopoietic progenitors are receptive to BCR-ABL expression and generate an abnormal expansion of mature myeloid cells, a clinical feature of CML in chronic phase. 26,27 These results demonstrate that although the Ph ϩ originates in HSCs, multipotent hematopoietic progenitors and not HSCs are the cell population responding to BCR-ABL in CML.Numerous signaling pathways regulated by BCR-ABL expression have been identified over the past decade; however, their relevance toward the direct effect of BCR-ABL expression in multipotent hematopoietic progenitors remains unclear for 2 reasons. First, most BCR-ABL studies have been conducted in fibroblast, myeloid, and pro-B cell lines, which do not represent multipotent hematopoietic progenitors. 28 Second, BCR-ABL studies conducted in the cellular context of hematopoie...

Chronic myeloid leukemia is effectively treated with imatinib, but reactivation of BCR-ABL frequently occurs through acquisition of kinase domain mutations. The additional approved ABL tyrosine kinase inhibitors (TKIs) nilotinib and dasatinib, along with investigational TKIs such as ponatinib (AP24534) and DCC-2036, support the possibility that mutationmediated resistance in chronic myeloid leukemia can be fully controlled; however, the molecular events underlying resistance in patients lacking BCR-ABL point mutations are largely unknown. We previously reported on an insertion/ truncation mutant, BCR-ABL 35INS IntroductionImatinib is an inhibitor of BCR-ABL, the tyrosine kinase that causes chronic myeloid leukemia (CML). Most newly diagnosed patients achieve durable remissions on imatinib therapy, 1,2 but 10%-15% fail to respond or relapse. The leading cause of imatinib resistance is reactivation of BCR-ABL because of kinase domain point mutations. Most BCR-ABL mutants are susceptible to alternative ABL tyrosine kinase inhibitor (TKI) therapies. [3][4][5][6][7][8] Sequencing of the BCR-ABL kinase domain in patients exhibiting signs of TKI treatment failure has also revealed the presence of alternatively spliced variants, including BCR-ABL 35INS , in which retention of 35 intronic nucleotides at the exon 8/9 splice junction introduces a stop codon after 10 intron-encoded residues. 9-13 The result is loss of the last 653 residues of BCR-ABL, including 22 native kinase domain residues. 10,12 Notably, the reported frequency of detection of the BCR-ABL 35INS mutant in cases of imatinib resistance (including instances in which a point mutation is concurrently detected in the BCR-ABL kinase domain) as detected by direct sequencing is ϳ1%-2%, 10,14 although more sensitive quantitative assays have reported detection of very low levels of the mutant transcript at a considerably increased prevalence. 14 Although BCR-ABL truncated immediately after the ABL kinase domain is fully transforming in a murine model of CML, 15 we predicted BCR-ABL 35INS would lack kinase activity, because the mutation eliminates the last 2 helices of the ABL kinase domain and disrupts a complex set of interactions among noncontiguous residues. 10 By contrast, recent reports have suggested that BCR-ABL 35INS confers TKI resistance in CML 9,12,14,16 and have proposed a BCR-ABL 35INS tailored clinical trial, 16 but they have not addressed the mechanism for this or assessed BCR-ABL 35INS catalytic activity. We provide cell-based and biochemical studies of BCR-ABL 35INS and a retrospective analysis of its detection in the context of treatment and response in CML patients. Methods IL-3 withdrawalBa/F3 cells cultured in standard media (RPMI 1640 media, 10% FBS, L-glutamine, penicillin-streptomycin; Invitrogen) containing IL-3 from WEHI-conditioned media were infected with retrovirus expressing BCR-ABL, BCR-ABL 35INS , or BCR-ABL K271P/35INS (MSCV-IRES-GFP), and stable cell lines were sorted for GFP (FACSAria II; BD Biosciences). (E) Equimolar amounts...