Chronic myelogenous leukemia (CML) was first described

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1 Imatinib Resistance Obstacles and Opportunities Mark R. Litzow, MD Objective. To review the current status of resistance to imatinib mesylate (IM) in patients with chronic myelogenous leukemia, and the obstacles and opportunities presented by the development of this resistance. Data Sources and Study Selection. Review of selected studies obtained from a MEDLINE search encompassing the years 1950 to Data Extraction and Data Synthesis. Relevant information from the selected studies was abstracted and summarized. Conclusions. The identification of the Philadelphia chromosome and the subsequent discovery that it represents a translocation between the long arms of chromosomes 9 and 22 producing an aberrant tyrosine kinase, known as BCR-ABL1, has catalyzed our understanding and treatment of this hematologic malignancy. An extensive search for molecules to block the aberrant BCR-ABL1 protein resulted in the development of IM as an orally bioavailable agent with remarkable efficacy in producing hematologic, cytogenetic, and molecular remissions. However, follow-up of patients treated with IM has demonstrated that some patients can develop resistance to IM with progression of their leukemia. Multiple mechanisms of resistance have been identified. The dominant mechanism appears to be mutations in the kinase domain of BCR- ABL1, which result in altered affinity of IM for the BCR- ABL1 protein. Recently, small-molecule, combined SRC and ABL1 inhibitors have been developed and entered into clinical trials. These inhibitors appear effective in inhibiting most of the mutant BCR-ABL1 molecules that are resistant to IM. The rapid development of new therapies for treatment of chronic myelogenous leukemia brings the promise that this disorder can be cured or controlled in many patients with oral drugs that have a low toxicity profile. (Arch Pathol Lab Med. 2006;130: ) Chronic myelogenous leukemia (CML) was first described as a distinct clinical entity less than 160 years ago. 1 However, it was not until 1960, when Nowell and Hungerford 2 described a minute acrocentric chromosome in the cells of 7 patients with CML, that the modern era of our understanding and treatment of CML began. During the past 45 years, tremendous advances have been made in the understanding of the pathogenesis and treatment of this chronic hematologic malignancy. These advances have recently culminated in the emergence of imatinib mesylate (IM), the first rationally designed, molecularly targeted therapy for a human malignancy. 3 It was not until 1973, however, that the minute chromosome identified by Nowell and Hungerford 2 was recognized to result from a reciprocal translocation between chromosomes 9 and 22. This translocation has now been designated as t(9;22)(q34;q11). 4 By the mid 1980s, it became apparent that this reciprocal translocation resulted in the Accepted for publication December 1, From the Mayo Clinic College of Medicine, Mayo Clinic, Rochester, Minn. The author has no relevant financial interest in the products or companies described in this article. Presented at William Beaumont Hospital s 13th Annual Symposium on DNA Technology in the Clinical Laboratory, Troy, Mich, October 7 9, Reprints: Mark R. Litzow, MD, Mayo Clinic College of Medicine, Mayo Clinic, 200 First St, SW, Rochester, MN ( litzow. mark@mayo.edu). juxtaposition of the Abelson (ABL1) oncogene, a tyrosine kinase on chromosome 9 to the breakpoint cluster region (BCR), a gene of unknown function on the long arm of chromosome The presence of the BCR-ABL1 gene rearrangement in virtually all cases of CML provides a unique molecular signature for this disease. Its identification has been of significant benefit in enhancing the diagnosis and monitoring of outcomes of therapy. The essential role of BCR-ABL1 in the pathogenesis of CML has been proven by the development of a CML-like syndrome in mice whose bone marrow was transfected with a BCR- ABL1 gene and transplanted into an irradiated syngeneic recipient. 6 The breakpoint in the ABL1 gene usually occurs in a fairly restricted portion of exon 2 of ABL1, whereas the breakpoint on the BCR gene can occur in multiple regions and result in different-sized hybrid aberrant tyrosine kinase genes. The most common breakpoint occurs in the major breakpoint cluster region (M BCR) of the BCR gene between exons 12 and 16 (also known as b1 to b5) and results in a 210-kd gene known as the p210 BCR-ABL. The chimeric protein that results is the one most frequently seen in typical cases of CML. Breakpoints in BCR occurring in the minor BCR (m-bcr) region splice out exons e1 and e2 and form a smaller, 190-kd BCR-ABL1 known as p190 BCR-ABL. This is seen most commonly in Philadelphia chromosome (Ph) positive acute lymphoblastic leukemia. 7 A third breakpoint has been noted in the e19 and e20 Arch Pathol Lab Med Vol 130, May 2006 Imatinib Resistance Litzow 669

2 exons and is known as the -BCR, which produces a 230- kd protein known as p230 BCR-ABL1. The clinical disorder seen with this latter e19a2 transcript is a Ph-positive chronic neutrophilic leukemia 7 (Figure 1, A and B). It is thought that CML arises from a pluripotent hematopoietic stem cell that has acquired the BCR-ABL1 gene rearrangement and allows it to gain a growth advantage over normal hematopoietic cells with suppression of normal hematopoiesis. 8 The CML progenitor cells exert their dominance over normal hematopoietic cells not only because of a proliferative advantage but also as a result of prolonged survival resulting from decreased apoptosis and altered adherence to marrow stromal elements, which appear to facilitate the release of CML progenitors into the peripheral blood. 9,10 The structural and functional relationships of BCR-ABL1 have been evaluated extensively. An interesting difference between BCR-ABL1 and its normal counterpart, the ABL1 protein, is their contrasting subcellular locations. The BCR- ABL1 is exclusively found in the cytoplasm, where it is constitutively activated. The ABL1 protein is found in both the nucleus and the cytoplasm and can shuttle between these 2 locations. 11 The SRC-homology 1 (SH1) domain of the ABL1 component of BCR-ABL1 appears to be the most crucial for malignant transformation. In ABL1, the SRC-homology 2 (SH2) domain and the C-terminal actin-binding domains fulfill important functions, and in the BCR portion of the molecule, the coil-motif encoded by the first bcr exon is responsible for dimerization of the BCR-ABL1. 12 The BCR-ABL1 protein exerts its proliferative potential by phosphorylating tyrosine on a large number of substrate molecules. 12 However, the function of these substrates and their role in leukemogenesis remains unclear (Figure 2). CLINICAL COURSE AND THERAPY Chronic myelogenous leukemia, if not effectively treated, follows a triphasic clinical course with an initial chronic phase that has an average duration of 5 to 6 years and is characterized by a mature leukocytosis and/or thrombocytosis with immature myeloid elements in the blood and frequent basophilia. Eventually, CML can transform to an acute leukemic phase that can be either myeloid or lymphoid in phenotype. This so-called blast phase can sometimes be controlled transiently with chemotherapy, but usually results in the eventual demise of the patient. One frequently sees an intermediate phase, known as the accelerated phase, between the chronic and blast phases; this phase appears when response to conventional therapy is lost and additional chromosome abnormalities may be seen in the bone marrow. 7 However, not all patients follow this triphasic course; some progress from the chronic phase directly to the blast phase and others die of complications in the chronic or accelerated phase. Up until the 1980s, CML typically followed an inexorable course that resulted in the death of the patient. Different scoring systems have been developed that have been able to categorize patients into groups based on disease characteristics and with varying projected lengths of survival. The most prominent of these systems is the one developed by Sokal et al 13 in the 1980s. Currently, only allogeneic hematopoietic stem cell transplantation has been shown conclusively to provide longterm disease eradication and prolonged disease-free survival for patients with CML. 14 Subsequent studies have confirmed the durability of disease response seen with transplantation and demonstrated that the use of alternative stem cell sources, such as unrelated donors, can provide outcomes in selected patients that are similar if not equal to those seen with matched related donors. Results of allogeneic transplantation for CML have recently been summarized. 15 However, availability of allogeneic transplantation to patients with CML is limited by the availability of suitable donors and concerns about treatment-related morbidity and mortality as a result of acute and chronic graft-versus-host disease, infection, and organ toxicity. Thus, transplantation has generally been limited to younger patients with good organ function and performance score. Accordingly, it has been estimated that only a small minority of patients with CML are eligible for conventional allogeneic transplantation. The less-intensive, nonmyeloablative stem cell transplant approach to CML has broadened the availability of this treatment modality to older patients and patients with compromised organ function. 16 Even with this approach, though, many patients still will not be candidates for or elect to proceed with allogeneic transplantation, and alternative forms of therapy for these patients have been sought through the years. The use of oral chemotherapy drugs such as busulfan and hydroxyurea have been shown to control blood counts and improve symptoms in patients with CML, but the impact of these drugs on improving survival has been minimal. 17 The isolation and subsequent cloning of interferon- (IFN- ), a cellular glycoprotein, and the recognition of its antiproliferative and immunoregulatory effects led to trials of its use in patients with CML. Pilot studies and subsequent phase 2 studies confirmed the ability of IFN- to produce complete hematologic responses in up to 80% of patients with chronic-phase CML. More remarkably, marked reductions were seen in the percentage of Ph-positive cells in cytogenetic analysis in the marrow of treated patients. Patients achieving a major cytogenetic response ( 33% Ph-positive cells) or complete cytogenetic responses (0% Ph-positive cells) appeared to have prolonged survival compared with patients with lesser responses. 18 These encouraging results have led to multiple randomized trials comparing IFN- with hydroxyurea and busulfan, and a meta-analysis of these studies clearly demonstrated improved survival with IFN- compared with chemotherapy. Five-year survival rates of 57% with IFN- and 42% with chemotherapy were reported. 19 Subsequent studies comparing a combination of IFN- with low-dose cytosine arabinoside demonstrated improved response rates with the combination, and improved survival in one but not the other study with the combination regimen compared with IFN- alone. The difference in survival outcome may be related to study design. 20,21.However, IFN- is associated with significant toxicity in some patients and has the inconvenience of subcutaneous injection. 22 The most exciting breakthrough in the treatment of CML has been the development of IM as an orally bioavailable therapeutic agent. The identification of the presence of BCR-ABL1 in the majority of patients with CML and the dependence of BCR-ABL1 function on its tyrosine kinase activity made it an attractive target in the search for selective kinase inhibitors. Imatinib mesylate, originally designated signal transduction inhibitor 571 (STI571), 670 Arch Pathol Lab Med Vol 130, May 2006 Imatinib Resistance Litzow

3 Figure 1. A, Schematic representation of the t(9;22)(q34;q11) or Philadelphia translocation. On the derivative chromosome 22 (Philadelphia chromosome [Ph]), BCR sequences are fused upstream of ABL1 sequences. The reciprocal fusion is formed on the derivative chromosome 9q. B, Molecular structure of the BCR-ABL1 translocation. Although the breakpoints in ABL1 are spread over a wide genomic region, they tend to cluster within BCR. In almost all patients with CML and approximately one third of patients with Ph-positive acute lymphyocytic leukemia, the breaks occur in the major breakpoint cluster region (M BCR), giving rise to e13a2 and e14a2 fusion mrnas (formerly referred to as b2a2 and b3a2, respectively) that are translated into a 210-kd protein. In two thirds of patients with Ph-positive acute lymphyocytic leukemia, the break in BCR occurs more 5, leading to an e1a2 fusion mrna and a 190-kd protein. Breakpoints between BCR exons 19 and 20 are found in many patients with chronic neutrophilic leukemia. Irrespective of the specific position of the break in ABL1, from alternative splicing, all fusions contain BCR sequences fused to ABL1 exon 2. C, Structural domains in ABL1. The N-terminus contains the 2 alternative first exons, 3 SRC homology (SH) domains, most importantly the SH1 domain that has tyrosine kinase activity. Only ABL1 exon Ib is myristoylated (myr). The long C terminus distinguishes ABL1 from other nonmembrane tyrosine kinases. It contains proline-rich regions (PxxP) that bind SH3 domains of other proteins, as well as DNA-binding domains (DNA BD), nuclear localization signals (NLS) and a nuclear export signal (NES). The C terminus is also responsible for binding to actin. Shown are also phosphorylation sites for protein kinase C (PKC), ataxia teleangiectasia muted (ATM), cdc2 as well as tyrosine 393 (Y393), the major regulatory tyrosine in the activation loop of the kinase. Reprinted with permission from J Cancer Res Clin Oncol. 89 Arch Pathol Lab Med Vol 130, May 2006 Imatinib Resistance Litzow 671

4 Figure 2. Signal-transduction pathways affected by BCR-ABL. The cellular effects of BCR-ABL1 are exerted through interactions with various proteins that transduce the oncogenic signals responsible for the activation or repression of gene transcription, mitochondrial processing of apoptotic responses, cytoskeletal organization, and of the degradation of inhibitory proteins. The key pathways implicated so far are those involving Ras, mitogen-activated protein (MAP) kinases, signal transducers and activators of transcription (STAT), phosphatidylinositol 3-kinase (PI3K), and Myc. Most of the interactions are mediated through tyrosine phosphorylation and require the binding of BCR-ABL1 to adapter proteins such as growth factor receptorbound protein 2 (GRB-2), DOK, CRK, CRKlike protein (CRKL), SRC-homology-containing protein (SHC), and casitas-b-lineage lymphoma protein (CBL). Reprinted with permission from N Engl J Med. 12 arose from a time-consuming process of random screening of large numbers of compounds. Imatinib mesylate is a 2-phenyl-amino-pyrimidine and it emerged as one of the most potent substances inhibiting the ABL protein. It also inhibits other kinases, predominantly those related to platelet-derived growth factor receptors and c-kit. 3 Its favorable oral bioavailability profile and lack of significant toxicity in animal models led to the design of large phase 1 and 2 trials to test its safety and efficacy in humans. The initial phase 1 trials of this agent were reported in 2001 and demonstrated that doses could be successfully escalated from 25 mg/d up to a high of 1000 mg/d. Adverse side effects from IM were minimal, with the most common being nausea, myalgias, edema, and diarrhea. In fact, a maximum tolerated dose could not be identified as a result of these studies. The initial trial included 83 patients with chronic-phase CML who had failed treatment with IFN-. Complete hematologic responses were seen in 53 of 54 patients who were treated with IM in doses of 300 mg/d or more. Of these 54 patients, cytogenetic responses occurred in 29. Seventeen patients achieved major cytogenetic responses and 7 had a complete cytogenetic remission. 23 These results were truly remarkable for this group of patients. A second phase 1 trial was performed in 58 patients with myeloid (38 patients) or lymphoid (20 patients, including Ph-positive acute lymphoblastic leukemia) blast crisis of CML. These patients received doses ranging from 300 to 1000 mg/d. Fifty-five percent (21/38) of the patients with myeloid blast crisis demonstrated responses; 4 of these 21 patients had a complete hematologic response. Seventy percent (14/20) of the patients with lymphoid blast crisis or Ph-positive acute lymphoblastic leukemia had a response, including 4 who had a complete hematologic response. Seven patients with myeloid blast crisis were continuing to receive treatment at the time of the report. All but 1 of the patients with lymphoid blast crisis or Ph-positive acute lymphoblastic leukemia who responded had relapsed. Frequent adverse effects in these patients were nausea, vomiting, edema, neutropenia, and thrombocytopenia. 24 These studies led to Federal Drug Administration approval in May 2001 of IM at a dose of 400 mg/d. Subsequent larger phase 2 studies confirmed the clinical benefit of IM. A study of 532 patients with late chronicphase CML who had failed prior IFN- therapy were evaluated for treatment with oral IM at a dose of 400 mg/d. Ninety-five percent of these patients achieved a complete hematologic response; a major cytogenetic response was achieved in 60%. Eighty-nine percent of patients did not progress to accelerated or blast phase of CML after 18 months of follow-up, and only 2% of patients discontinued treatment because of adverse side effects. 25 A long-term follow-up of 261 of these patients (with a median followup of nearly 4 years) showed a major cytogenetic response in 73% of the patients and a complete cytogenetic response in 63%. At 4 years, the percentage of patients achieving a major molecular response by quantitative reverse transcriptase polymerase chain reaction, with a BCR-ABL1/ABL1 ratio less than 0.05%, was 43%, and a complete molecular response (BCR-ABL1 undetectable) occurred in 26%. 26 Results in a similarly treated group of patients with chronic-phase CML who had failed IFN- therapy have been reported from Europe. 27 A phase 2 study of IM at doses of 400 or 600 mg/d in patients with accelerated-phase CML showed encouraging, but not as significant, results compared with patients in the chronic phase. Hematologic responses were seen in 82% of patients, with a major cytogenetic response of 24% (complete in 17%). In this group of patients, doses of 600 mg/d demonstrated an improved cytogenetic response of 28% compared with 16% in the patients who were treated with doses of 400 mg/d. Duration of response, time to disease progression, and survival were also improved in patients receiving the higher dose. 28 Similar phase 2 studies in patients with myeloid and lymphoid blast crisis or Ph-positive acute lymphoblastic leukemia with doses of IM of 400 or 600 mg/d again showed impressive responses. Patients in these advanced phases demonstrated lower responses compared with those of patients treated in the chronic or accelerated phase. For myeloid blast crisis, hematologic responses 672 Arch Pathol Lab Med Vol 130, May 2006 Imatinib Resistance Litzow

5 n Table 1. Results of Phase II Trials of Imatinib in Patients With Chronic Myelogenous Leukemia or Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia* Disease Phase CP CP AP MBP LBP Prior Therapy HR MCR CCR Reference IFN- IFN- Various Various NS * HR indicates hematologic response; MCR, major cytogenetic response; CCR, complete cytogenetic response; CP, chronic phase; IFN-, interferon- ; AP, accelerated phase; MBP, myeloid blast phase; LBP, lymphoid blast phase; and NS, not significant. Complete or partial hematologic responses. were seen in 52% of patients, with 8% of the responses being complete. Major cytogenetic responses were seen in 16% of patients, with 7% of the responses being complete. 29 For patients with lymphoid blast crisis, complete hematologic responses were seen in 19%, with complete cytogenetic responses in 17%. 30 See Table 1 for a summary of these phase 2 studies. These overall encouraging results led to a large randomized trial comparing IM at 400 mg/d with IFN- plus low-dose cytarabine in patients with newly diagnosed chronic-phase CML, the International Randomized Trial of Interferon and STI571 (IRIS). In this trial there were 1106 patients, 553 in each arm. Crossover to the alternative therapy was allowed. The initial report of this study, with a median follow-up of 19 months, showed that the estimated rate of major cytogenetic response was 87% in the IM group and 35% in the IFN- plus cytarabine group. Complete cytogenetic response was achieved in 76% of patients who were given IM, compared with only 15% in the IFN- plus cytarabine group. Only 15% of the patients in the IM group, compared with 89% in the IFN- plus cytarabine group, discontinued treatment or crossed over to the alternative treatment group. Intolerance of the IFN- plus cytarabine combination was the most common reason for crossover. 31 A recent update of the study has been published in abstract form. 32 At 30-month follow-up, the complete hematologic response for the IM group was 95%, the major cytogenetic response rate was 87%, and the complete cytogenetic response rate was 79%. The estimated percentage of major molecular response at 12 months was 40%. Freedom from progression at 30 months was 88%; freedom from progression to accelerated or blast phase was 95%, and the estimated survival at 30 months was also 95%. 32 A subsequent publication assessing the major molecular responses in patients in this study using the quantitative real-time polymerase chain reaction assay has demonstrated that 57% of the patients in the IM group who achieved a complete cytogenetic response had at least a 3-log reduction in BCR-ABL1 transcript, compared with 24% of those with a complete cytogenetic response in the IFN- plus cytarabine group. Patients with a complete cytogenetic response and at least a 3-log reduction in BCR-ABL1 transcript at 12 months have a 100% probability of remaining progression-free at 24 months, compared with 95% of patients with less than a 3-log reduction and 85% for patients who are not in complete cytogenetic remission at 12 months (P.001). 33 Figure 3. Proportion of patients lacking or losing hematologic response within 2 years of imatinib therapy. CP indicates chronic phase; AP, accelerated phase; My. BC, myeloid blast crisis. Reprinted with permission from Hematol Oncol Clin North Am. 34 Table 2. Definitions of Resistance* Primary (intrinsic) Secondary (acquired) Subcategories of resistance Hematologic Chronic phase Advanced phases Cytogenetic Molecular * See text for detailed explanations. These studies, however, also demonstrated that varying proportions of patients did not achieve clinical responses to IM therapy. In the IRIS trial after a median follow-up of 19 months, approximately 5% of patients did not achieve a complete hematologic remission. In accelerated and blast phases of CML, higher proportions of patients do not achieve complete hematologic response or subsequent cytogenetic response 28,29 (see Figure 3). Thus, these patients would be defined operationally as resistant to IM therapy. IM RESISTANCE Paralleling the wide application of IM to the treatment of patients with CML was the evolution of studies exploring the mechanisms of resistance. Before outlining these mechanisms, it is important that the varying types of resistance to IM that can occur are clearly defined. These resistance definitions are outlined in Table 2. Intrinsic or primary resistance to IM is when there is a lack of hematologic or cytogenetic response to initial IM therapy. Acquired or secondary resistance or relapse is a loss of benefit of IM after an initial response to therapy. Either of these types of resistance can be subdivided into hematologic, cytogenetic, or molecular resistance. Hematologic resistance is further subdivided by the phase of CML to which one is referring. For example, in the chronic phase, hematologic resistance refers to a loss or lack of normalization of spleen size, peripheral blood counts, or differential. Accelerated or blast phase of hematologic resistance indicates a lack of return to the chronic phase or Arch Pathol Lab Med Vol 130, May 2006 Imatinib Resistance Litzow 673

6 Table 3. Mechanisms of Imatinib Resistance Decreased intracellular drug levels Plasma binding by -1 acid glycoprotein Drug efflux from P-glycoprotein (MDR-1) overexpression Increased expression of BCR-ABL kinase from genomic amplification Clonal evolution (non-bcr-abl dependent mechanism) Mutations in ABL kinase of BCR-ABL affecting drug interaction or kinase activity a hematologic relapse following an initial response to therapy. Resistance at the cytogenetic level can occur when there is a loss or a lack of major ( 34%) Ph-positive metaphases or complete (0%) Ph-positive metaphases remission at the cytogenetic level. Resistance at the molecular level indicates a loss or lack of complete molecular response, such as undetectable BCR-ABL1 transcripts by either a real-time quantitative polymerase chain reaction, or the loss or lack of a major molecular remission, which is defined as a more than 3- log reduction of BCR-ABL1 transcripts or a BCR/BCR- ABL1 ratio less than 0.1%, respectively. These definitions of resistance are outlined in an excellent review article on this topic by Hochhaus and Hughes. 34 Multiple mechanisms of resistance to IM have been defined in the last several years. Four broad mechanisms of IM resistance have been characterized (Table 3). Several of these were predicted by in vitro studies modeling resistance. Human cell lines that are BCR-ABL1-positive and murine hematopoietic cells that have been transformed with the BCR-ABL1 gene have been exposed to IM with subsequent development of resistance. 35 These models have predicted several of the mechanisms of IM resistance demonstrated in vivo and outlined here. A mouse model of IM resistance has shown that although in vivo tumors were resistant to IM, they retained in vitro sensitivity to IM. 36 Clinical studies have suggested that plasma binding of -1 acid glycoprotein correlated with clinical responses to IM, but none of the studies clearly distinguished cause and effect. 37,38 In vitro models have suggested that expression of the multidrug resistance P-glycoprotein could contribute to IM resistance. 35,39 Leukemic cells from patients with positive results for P-glycoprotein were correlated with a decrease in intracellular IM levels and resistance, and suggested that IM may be sensitive to drug efflux via P-glycoprotein. 40 A more recently identified potential novel mechanism for IM resistance is the downregulation of T-cell protein tyrosine phosphatase, which has been shown in vitro to be downregulated in IM-resistant cells. 41 Two studies have demonstrated that gene amplification of the BCR-ABL1 kinase is associated with development of IM resistance. This has been demonstrated by the presence of multiple copies of the BCR-ABL1 gene in interphase nuclei by the use of a fluorescence in situ hybridization assay. 42,43 In one of these studies, more than half the patients with IM resistance had evidence of clonal evolution with the development of novel chromosome abnormalities in addition to the p(9;22)(q34;q11). This evolution was observed in paired cytogenetic analyses noted at the beginning of IM therapy and at the time of resistance. These chromosomal abnormalities included aneuploidy, a second Ph chromosome in 8 patients, and trisomy 8 in another 6 patients. The loss of one p53 allele via alteration of the short arm of chromosome 17 was seen in 7 patients and the new reciprocal translocations were seen in 2 patients. In 8 cases, the presence of multiple cytogenetic abnormalities was also noted. 43 The dominant and best well-studied mechanism of resistance to IM in patients with CML are gene mutations in the ABL1 (tyrosine kinase) domain of the BCR-ABL1 gene. The first such mutation was described in 2001 by Gorre et al, 42 in which a single amino acid substitution in a threonine residue of ABL1 kinase domain resulted in substitution of isoleucine (T315I) for threonine. This single amino acid substitution interfered with a critical hydrogen bond that forms between the ABL1 kinase and IM. This altered binding prevented IM inhibition of BCR-ABL1 and conferred resistance to IM in vitro. It appears to be a significant mechanism of in vivo resistance because of the frequent finding of the T315I in many patients with IM resistance. It is important to note that the c-abl1 protein is expressed in 2 splice forms known as 1a and 1b. The 1b form is 19 residues longer than the 1a form and contains a myristoylation site on its second residue. This second residue, which is a glycine, helps regulate enzymatic activity, and its mutation to alanine prevents myristoylation and results in an activated kinase. 44 The shorter 1a form of c-abl1 may be regulated by a similar mechanism. The type 1b contains a cap region that is believed to stabilize the inactive conformation of the kinase. 45 The most commonly used numbering system to identify the amino acid residues where mutations have occurred in the ABL1 kinase are based on the shorter 1a form of the ABL1 kinase. However, the numbering from the 1b form is becoming the accepted convention for describing the location of mutations. Therefore, the threonine-isoleucine change at position 315 of the type 1a form would be at position 334 of the type 1b form. It is important to have some rudimentary understanding of the structure of the BCR-ABL1 chimeric protein to understand how the various mutations in the ABL1 kinase domain confer a resistance to IM (see Figure 1, C). Both the 1a and 1b spliced variants of c-abl1 have 3 SRC homology domains, including SH3, which is a negative regulator of kinase activity; SH2, which binds peptides that contain phosphotyrosine; and SH1, the kinase domain, which encodes for catalytic function. At the N-terminal end of the ABL1 kinase is a highly conserved nucleotidephosphate binding domain for adenosine triphosphate, which is known as the. At the carboxy or C-terminal end of the molecule is a flexible activation loop that is essential for the control of catalytic activity and changes conformation depending on whether the molecule is in the inactive or active state. Between these 2 loops is the catalytic site of the molecule, which resides in a cleft where IM and other small molecule tyrosine kinase inhibitors bind. Both ABL1 and SRC shift between an inactive or a closed conformation and a catalytically active or open conformation, and this shift from an inactive to active state appears to be regulated by the kinase itself in a process known as autoinhibition. In the inactive state, the activation loop just described is folded inward toward the catalytic site, and in the active state, this activation loop flips away from the catalytic region. Thus, it can act as a support for substrate binding. These interactions are more 674 Arch Pathol Lab Med Vol 130, May 2006 Imatinib Resistance Litzow

7 Table 4. Mutations Mutations and Their Location Within the Kinase* ABL Type 1a ABL Type 1b Location in BCR-ABL M 244 V L 248 V G 250 E Q 252 H Q 252 R Y 253 H Y 253 F E 255 K E 255 V V 289 A F 311 T T 315 I F 317 L M 343 T M 351 T E 355 G F 359 V V 379 I F 382 L L 387 M H 396 P H 396 R S 417 Y E 459 K F 486 S M 263 V L 267 R G 269 E Q 271 H Q 271 R Y 272 H Y 272 F E 274 K E 274 V V 308 A F 330 T T 334 I F 336 L M 362 T M 370 T E 374 G F 378 V V 398 I F 401 L L 406 M H 415 P H 415 R S 4436 Y E 478 K F 505 S (nucleotide binding loop) Active site (imatinib contact site) Active site Active site Active site SH2 contact SH2 contact C-terminal lobe Active site A-loop (activation loop) A-loop A-loop A-loop A-loop C-terminal lobe C-terminal lobe C-terminal lobe * Reprinted with permission from Curr Opin Hematol. 46 thoroughly reviewed by Nardi et al, 46 and described in recent publications. 45,47 Mutations within the ABL1 kinase domain are being increasingly described; to date, more than 30 such mutations have been identified. The more common and best-defined mechanisms of resistance to IM involve a diverse number of mutations occurring in the tyrosine kinase domain of the BCR-ABL1 gene. These single nucleotide substitutions result in substitution of single amino acids that have varying effects on the conformation of the ABL1 portion of the BCR-ABL1 and its binding to drugs or substrates. Studies exploring the crystal structure of the catalytic domain of ABL1 when complexed to IM predicted that this mutation would result in resistance because the threonine residue at position 334 is known to form a crucial hydrogen bond between the ABL1 kinase and IM. 48 From these studies it also became clear that IM binds BCR-ABL1 in its inactive conformation only. The mutations in the ABL kinase domain are listed in Table 4 and their frequency is shown in Figure 4. 34,49 As can be seen from Table 3 and Figures 4 and 5, these mutations are distributed at multiple sites throughout the BCR-ABL1 kinase domain, including within the nucleotide binding or, within the active site where IM binds and within the activation loop and the carboxy terminal. Mutations within the are among the most common. 34,46 In vitro studies in which the BCR-ABL1 molecule is randomly mutated and screened for IM resistance were able to recover the major mutations that had been identified in patients up to the time of publication of this study, but also numerous other mutations that illustrate potentially novel mechanisms of acquired IM resistance. 50 The ability of these mutated BCR-ABL1 proteins to confer IM resistance has been confirmed by their association with clinical resistance in patients and the inability of IM to inhibit proliferation of cell lines transfected with the various BCR-ABL1 mutants. 51 Of particular interest is the fact that 2 of the major mutations that confer IM resistance, known as E274K and the Figure 4. Location of BCR-ABL1 mutations within the kinase domain. Fifty-nine patients had mutations detected at 23 different sites. Fourteen patients had 2 to 4 mutations. This analysis is based on a survey of 248 consecutive patients treated with imatinib. Reprinted with permission from Hematol Oncol Clin North Am. 34 Arch Pathol Lab Med Vol 130, May 2006 Imatinib Resistance Litzow 675

8 Figure 5. ABL1 and complex with imatinib (yellow). The location of mutations are highlighted along with the activation loop (green), nucleotide-binding loop (red), and catalytic loop (orange). strands are numbered, and helices are lettered according to the nomenclature used for insulin-receptor tyrosine kinase. The region including N322 and interacting with the nucleoside-binding loop is shown in cyan. Reprinted with permission from Lancet Oncol. 90 originally described T334I, appear to enhance the activity of the BCR-ABL1 kinase through an increased ability to induce autophosphorylation. These results suggest that these resistance mutations may confer a growth advantage for CML cells, even in the absence of selective pressure from treatment with IM. 52 These data would be consistent with the finding that mutations in the BCR-ABL1 gene that confer IM resistance can be identified in patients prior to treatment with IM. 53,54 By eradicating IM-sensitive cells, selective pressure could thus be exerted to allow expansion of IM-resistant clones following treatment. Thus, it appears that mutations conferring IM resistance fall into at least 2 significant groups: (1) those that change amino acids that are directly in contact with IM as one mechanism, and (2) mutations that appear to prevent BCR-ABL1 from achieving a conformational state that is inactive, which, as previously noted, is the required conformation for IM binding. 55 However, not all mutants are associated with resistance to IM. A recent study found that 5 of 17 tested ABL1 kinase domain mutants remained sensitive to IM. 56. Mutations within the nucleotide-phosphate binding domain for adenosine triphosphate (also known as the adenosine triphosphate phosphate-binding loop or ) appear to confer a poorer prognosis. In a study of 144 patients from Australia, mutations were detected in 27 patients at 17 different residues. Eighty-nine percent (24/27) of these patients developed acquired resistance. Of the 13 patients with mutations in the, 12 died, with a median survival of 4.5 months after the mutation was detected. Of the 14 patients with mutations outside the P- loop, there were only 3 deaths, with a median follow-up of 11 months. 57 This poor outcome for patients with mutations in the may relate to the enhanced activity of the BCR-ABL fusion kinase previously noted. 52 Studies from France and Italy have also suggested that patients with mutations have a poorer prognosis than those without this type of mutation. 58,59 However, another study from the United States was unable to confirm the poorer prognosis with mutations, so the exact significance of a mutation in terms of prognosis remains somewhat unknown. 60 As expected from the clinical data, mutations are more commonly found in patients in more advanced phases of disease and also in patients with additional chromosome abnormalities consistent with cytogenetic clonal evolution. 61 However, mutations as noted previously can be found in patients who are still in the chronic phase. 53 Gene expression profiles of bone marrow samples from patients with IM-sensitive and IM-resistant disease utilizing oligonucleotide microarray analysis have shown the ability to distinguish IM sensitivity and resistance. Data from these studies can be used to identify genes that are differentially expressed in patients with IM-sensitive and IM-resistant disease to identify new targets for therapy; these data also have the potential to be used as a screening tool to identify patients with resistance prior to therapy. 62,63 The ability to detect mutations in the BCR-ABL1 gene vary depending on the technique used. Methods vary from direct sequencing of the kinase domain, 57 use of denaturing high-performance liquid chromatography, 64 a flow cytometric measurement of downstream targets of BCR-ABL1, 65 restriction fragment length polymorphism based assays, 66 and various polymerase chain reaction based assays, including those involving allele-specific oligonucleotides, 53 peptide nucleic acid based clamping techniques, 67 identification of single nucleotide polymorphisms utilizing microarrays, 68 and real-time quantitative polymerase chain reaction. 69 The latter study has indicated that a 2-fold rise in BCR-ABL1 expression by real-time quantitative polymerase chain reaction predicted a mutation in the kinase domain of BCR-ABL1 in 61% of patients either at the time of rise in BCR-ABL1 expression or within 3 months. 69 OVERCOMING RESISTANCE The development of IM resistance in patients with CML has stimulated interest in defining therapeutic strategies to overcome resistance. Although it is not yet possible to routinely define the mechanism of resistance in each patient who fails therapy with IM, various strategies have been utilized to overcome this resistance. One of the most commonly utilized strategies to date is escalation of the dose of IM. This strategy was based on the efficacy of higher doses of IM in patients with accelerated and blast phases of CML, as previously discussed. 28,29 In patients with chronic-phase CML who are resistant to conventional doses of IM of 300 or 400 mg/d, escalation to twice-daily administration of these doses resulted in complete hematologic responses in 65% of patients with hematologic resistance. Patients with cytogenetic resistance achieved complete cytogenetic response 56% of the time Arch Pathol Lab Med Vol 130, May 2006 Imatinib Resistance Litzow

9 Methods to overcome some of the resistance mechanisms outlined here are of potential theoretical benefit but have not yet been tested clinically. These include blocking P-glycoprotein-mediated drug efflux 42 or treating patients with drugs that compete for -1 acid glycoprotein binding of IM, such as erythromycin. 71 Another unique strategy to overcome resistance that has not been thoroughly tested in the clinic is cessation or interruption of IM therapy. Anecdotal reports of withdrawal of IM therapy in a patient who developed blast phase while taking IM, with subsequent reversion to the chronic phase with withdrawal of IM, supports the potential clinical benefit of this approach. 72 The rationale behind this technique is that withdrawal of IM may allow the reappearance of leukemic clones that are not mutated and that can suppress the mutant clone by removing the competitive advantage of the mutant clone. In vitro and clinical studies have also attracted interest in the use of combination therapies to overcome IM resistance. This approach applies the rationale that combining drugs that are active against CML with differing mechanisms of action may allow either synergy or additive effects against the leukemic clone via a non cross-resistant mechanism. This field of inquiry has an extensive literature of its own and will not be extensively reviewed here. Hochhaus and La Rosee 73 have recently summarized this approach. The area that has attracted the most interest in overcoming IM resistance is the development of alternative small molecules that can inhibit the BCR-ABL1 kinase protein. A new class of compounds known as pyridopyrimidines, which was originally developed as SRC inhibitors, has recently been shown to inhibit wild-type ABL1 at nanomolar concentrations. 74 Testing of several of these compounds in vitro against IM-resistant BCR-ABL1 mutants demonstrated sensitivity of these agents However, these pyridopyrimidine derivatives were predicted to have unsatisfactory pharmacokinetic profiles that prevented their clinical development. Fortunately, alternative compounds to these agents have reached exciting stages of development. 78 The 2 compounds in the most active stages of development include BMS and AMN107. BMS is a carboxamide derivative that is a synthetic small-molecule inhibitor of the SRC family of kinases. A recent study has shown that this orally bioavailable BCR-ABL1 kinase inhibitor is active against 14 of 15 IM-resistant BCR-ABL1 mutants and has a 2-log increased potency relative to IM. 79 Only the T334I mutation has been shown to retain resistance to both BMS and the pyridopyrimidine derivatives derived, as previously mentioned. The difference in activity between IM and these more recently developed small-molecule inhibitors relates to several key structural elements of ABL1. Recall that the kinase activity of ABL1 is regulated by the position of the activation loop, which is a flexible structure that flips into different conformations in the inactive and active states of the kinase protein. Imatinib binds the ABL1 kinase domain in its inactive conformation with the activation loop in the closed position, as previously described. 48 The inactive conformations of ABL1 and SRC are distinct; therefore, IM is able to inhibit ABL1 but it is not able to inhibit SRC. In contrast, the pyridopyrimidine derivatives and BMS bind to ABL1 whether the activation loop is in the closed or open position (inactivated or activated). 80 Thus, binding is not influenced by the activation state. Additionally, these latter compounds are smaller than IM, so the must undergo major conformational changes on binding with IM, whereas only minimal changes occur with the smaller molecules. However, this also suggests that BMS and compounds like it may have broader activity against other kinases, including those in the SRC family, and have broader effects, including potentially adverse ones in clinical trials. Both BMS and AMN107 have entered phase 1 clinical trials. Preliminary results from these trials have been recently reported. Complete hematologic responses were seen in 86% of patients with IM resistance, with one third of patients who were treated for more than 3 months achieving a major or complete cytogenetic response, including 5 patients who had achieved complete cytogenetic responses. 81 Responses with BMS have also been seen in patients in accelerated and blast phases of CML. 82,83 In those patients achieving a major cytogenetic response, 1-log to 2-log reductions in BCR-ABL1 transcript by polymerase chain reaction were seen. BMS was not associated with significant side effects in these trials. Similarly, phase 1 and 2 testing of AMN107 has demonstrated complete hematologic responses with minimal toxicity in patients with IM-resistant CML. 84 An exciting new development has been the reporting of agents in preclinical development that are not adenosine triphosphate-competitive inhibitors of BCR-ABL1 and appear to be active against multiple BCR-ABL1 mutants, including the T334I mutation. 85 CONCLUSION The identification and evolving understanding of the structure and function of the pathognomonic BCR-ABL1 aberrant tyrosine kinase identified by the t(9;22) translocation has led to dramatic advances in our understanding of the pathogenesis of CML. This understanding has led to remarkable therapeutic advances, including the rational identification of IM, that are inhibitory of the BCR-ABL1. The dramatic responses of patients with CML and other BCR-ABL1 driven leukemias to IM and its lack of toxicity has been a stunning advance in the war on cancer. 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Blood. 2003;101: Branford S, Rudzki Z, Walsh S, et al. Detection of BCR-ABL mutations in patients with CML treated with imatinib is virtually always accompanied by clinical resistance, and mutations in the ATP phosphate-binding loop () are associated with a poor prognosis. Blood. 2003;102: Corm S, Nicollini F, Borie D, et al. Mutation status of imatinib mesylateresistant CML patients and clinical outcomes: a French multicenter retrospective study for the filmc group [abstract 275]. Blood. 2004;104:82a. 59. Soverni S, Martinelli G, Rosti G, et al. ABL mutations in late-chronic phase chronic myeloid leukemia patients with cytogenetic refratorieness to imatinib are associated with a greater likelihood of progression to blast crisis and shorter survival. On behalf of the GIMEMA working party on chronic myeloid leukemia [abstract 1005]. Blood. 2004;104:287a. 678 Arch Pathol Lab Med Vol 130, May 2006 Imatinib Resistance Litzow