STI571: Targeting BCR-ABL as Therapy for CML

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1 STI571: Targeting BCR-ABL as Therapy for CML MICHAEL J. MAURO, BRIAN J. DRUKER Leukemia Program, Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, Oregon, USA Key Words. Chronic myelogenous leukemia Tyrosine kinase Philadelphia chromosome STI571 ABSTRACT Therapeutic agent STI571 (signal transduction inhibitor number 571) is a rationally developed, potent, and selective inhibitor for abl tyrosine kinases, including bcr-abl, as well c-kit and the platelet-derived growth factor receptor tyrosine kinases. Results of clinical trials to date have demonstrated the crucial role of the bcr-abl tyrosine kinase in chronic myelogenous leukemia (CML) pathogenesis and the potential of anticancer agents designed to target specific molecular abnormalities in human cancer. An initial phase I study of STI571 included 83 Ph + CML patients who had failed interferon-based therapy. Patients were required to be in chronic phase, defined liberally as less than 15% blasts in blood or bone marrow. Patients were treated with once-daily oral doses of STI571 in 14 successive dose cohorts ranging from 25-1,000 mg. In this phase I study, no dose-limiting toxicity was encountered and toxicity at all dose levels was minimal. The threshold for a maximally effective dose was found at 300 mg; for patients treated at or above this level, complete INTRODUCTION Chronic myelogenous leukemia (CML) is a clonal hematopoetic stem cell disorder with an annual incidence of one to two cases per 100,000 per year. It progresses through distinct phases: the stable or chronic phase, advanced phases, the accelerated phase, and blast crisis. The chronic phase is characterized by massive expansion of myeloid cells, which maintain normal maturation. In the later phases, leukemic cells lose their capacity to terminally differentiate; the result is an acute leukemia, which is highly refractory to therapy. The cytogenetic hallmark of all phases of CML and a subset of cases of acute lymphoblastic leukemia (ALL) is the Philadelphia (Ph) chromosome. The Ph chromosome is a shortened chromosome 22 that results from a reciprocal translocation between the long arms of chromosomes 9 and hematologic response was seen in 98% of patients, with complete cytogenetic responses in 13% and major cytogenetic responses in 31%. With a median duration of followup of 310 days, ongoing responses are evident in 96% of patients. In the phase II study of the accelerated phase of CML, 233 patients were treated with either 400 or 600 mg of STI571. With similar follow-up to the chronic phase trial, 91% of patients showed a hematological response; 63% of patients achieved a complete hematological response but not all patients had recovery of peripheral blood counts. In addition to the phase II clinical trials with STI571, a phase III trial randomizing newly diagnosed patients to either interferon with low-dose s.c. cytosine arabinoside versus STI571 is ongoing; this trial accrued rapidly and data collection is ongoing. Integration of STI571 into CML treatment algorithms will require long-term followup data from the ongoing phase II and III clinical studies. The Oncologist 2001;6: The molecular consequence of this translocation is the fusion of the c-abl oncogene from chromosome 9 with sequences from chromosome 22, the breakpoint cluster region (bcr), giving rise to a fused bcr-abl gene. Depending on the site of the breakpoint in bcr, various different fusion proteins are produced: p185 (185 kda), p210 (210 kda), or rarely p230. The p210 protein is seen in 95% of patients with CML and up to 20% of adult patients with de novo ALL; the p185 form is seen in approximately 10% of patients with ALL and in the majority of pediatric patients with Ph + ALL (5% of all pediatric ALL cases). c-abl, the cellular homologue of the transforming protein found in the Abelson murine leukemia virus (v-abl), encodes for a nonreceptor tyrosine kinase. The c-abl protein has tightly regulated kinase activity and shuttles between the nucleus and Correspondence: Michael J. Mauro, M.D., Oregon Health Sciences University, OP28, 3181 SW Sam Jackson Park Road, Portland, OR 97201, USA. Telephone: ; Fax: ; maurom@ohsu.edu Received March 30, 2001; accepted for publication May 11, AlphaMed Press /2001/$5.00/0 The Oncologist 2001;6:

2 234 STI571: Targeting BCR-ABL as Therapy for CML cytoplasm, whereas the bcr-abl fusion proteins are exclusively cytoplasmic and have enhanced tyrosine kinase activity that is essential for their transforming ability. Transduction of bcr-abl into murine hematopoietic stem cells followed by transplantation into irradiated, syngeneic mice produces a CML-like syndrome, implicating bcr-abl as the causative molecular abnormality of CML and Ph + ALL. Tyrosine kinases are enzymes that transfer phosphate from ATP to tyrosine residues on substrate proteins that in turn regulate cellular processes such as proliferation, differentiation, and survival [1]. Therefore it is not surprising that deregulated tyrosine kinase activity has a central role in malignant transformation. This has also made tyrosine kinases attractive therapeutic targets for pharmacologic inhibition. Signal transduction inhibitor 571 (STI571; formerly CGP57148B), or Gleevec, is a rationally designed abl-specific tyrosine kinase inhibitor [1]. This article will review the development and clinical experience with STI571 and highlight ongoing and future efforts essential to optimally utilize this new therapeutic agent. DESIGN OF STI571 Since all protein kinases use ATP as a phosphate donor and as there is a high degree of conservation among kinase domains particularly in the ATP binding sites it was thought that inhibitors of ATP binding would lack sufficient target specificity to be clinically useful. However, in 1988, Yaish et al., reported the first synthetic tyrosine kinase inhibitors, known as tyrphostins, which demonstrated specificity among different tyrosine kinases [2]. Work done independently at Ciba-Geigy (now Novartis) using high throughput screening of compound libraries led to the identification of the 2-phenylaminopyrimidine class of kinase inhibitors. The initial compounds were of low potency and had poor specificity, but working from this lead compound, a series of related compounds were synthesized. Using structure-activity relationships, these compounds were optimized against a variety of targets. From this drug development program, STI571 was developed initially as a specific platelet-derived growth factor receptor (PDGF-R) inhibitor, but was also found to be a potent and selective inhibitor for abl tyrosine kinases, including bcr-abl (Fig. 1). PRECLINICAL INVESTIGATIONS In vitro studies in our laboratory revealed that the concentration (IC 50 ) of STI571 that resulted in a 50% reduction in substrate phosphorylation and cellular tyrosine phosphorylation induced by bcr-abl was µm and 0.25 µm, respectively [3]. Aside from the abl family of kinases and PDGF-R, the only other tyrosine kinase found to be inhibited by STI571 was c-kit. Further work showed that STI571 Figure 1. Schematic representation of the mechanism of action of STI571. The bcr-abl tyrosine kinase is a constitutively active kinase which functions by binding ATP and transferring phosphate from ATP to tyrosine residues on various substrates. This causes the excess proliferation of myeloid cells characteristic of CML. STI571 functions by blocking the binding of ATP to the bcr-abl tyrosine kinase, inhibiting its activity. In the absence of tyrosine kinase activity, substrates required for bcr-abl function cannot be phosphorylated and subsequent cellular events are abrogated.

3 Mauro, Druker 235 specifically inhibits the proliferation of myeloid cells containing p210 bcr-abl [3]. Colony-forming assays from CML patients, when incubated with 1 µm STI571, showed a marked decrease in the number of bcr-abl colonies without significant inhibition of normal colony formation. This work has been confirmed and extended by several laboratories. For example, long-term marrow cultures revealed that prolonged exposure over many weeks has a sustained inhibitory effect on CML progenitors with little toxicity to normal cells. Similar findings were found with investigation of p185 bcr-abl-bearing cell lines and patient samples as found in some cases of Ph + ALL; STI571 also inhibited the proliferation of Ph + ALL cell lines as well as fresh leukemic blast cells expressing the p185 fusion protein. Initial animal studies showed a dose-dependent inhibition of tumor growth in bcr-abl-inoculated mice treated daily with STI571, but in no case was the tumor eradicated. However, in subsequent experiments in nude mice, using a three-times-per-day dosing schedule, STI571 was successful in eradicating bcr-abl-containing tumors [4]. Recognizing that the half-life of STI571 is about 4 hours in mice led to the conclusion that continuous exposure to STI571 was required for optimal antileukemic effects. CLINICAL STUDIES The initial phase I study [5, 6] of STI571 included 83 Ph + CML patients who had failed interferon-based therapy. Patients were required to be in chronic phase, defined liberally as less than 15% blasts in blood or bone marrow. Patients were treated with once-daily oral doses of STI571 in 14 successive dose cohorts ranging from 25-1,000 mg. Important pharmacokinetic parameters were determined in this trial (Table 1). Mean plasma concentrations of 3.9 µg/ml (7.8 µm) are reached at steady state after a oncedaily dose of 600 mg. After rapid absorption following oral administration, STI571 is metabolized mainly by hepatic p450 enzymes, particularly the CYP3A4/5 isoenzymes. The terminal half-life of STI571 is hours, suggesting that once-daily administration is appropriate. Patients were treated at their originally assigned dose level unless disease progression or toxicity developed. In this Table 1. Pharmacokinetic parameters in CML patients at steady state following once daily oral administration of STI571 (values listed as mean ± SD) Dose Subjects T max C max T 1 / 2 AUC (0- ) (mg) (n) (h) (µg/ml) (h) (µg/h/ml) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± phase I study, no dose-limiting toxicity was encountered and toxicity at all dose levels was minimal. The threshold for a maximally effective dose was found at 300 mg; for patients treated at or above this level, complete hematologic response was seen in 98% of patients, with complete cytogenetic responses in 13% and major cytogenetic responses in 31%. With a median duration of follow-up of 310 days, ongoing responses are evident in 96% of patients. Based on its effectiveness in chronic phase patients who had failed interferon, the phase I studies were expanded to include CML patients in myeloid and lymphoid blast crisis and patients with relapsed or refractory Ph + ALL [7, 8]. Patients were treated with STI571 once daily, at doses of 300 to 1,000 mg. Twenty-four of 38 (55%) patients with myeloid blast crisis responded to therapy, defined by a decrease in percentage of marrow blasts to less than 15%; 8/38 (21%) had clearance of blasts from their marrows to less than or equal to 5%. Fourteen of 20 (70%) patients with lymphoid phenotype disease, CML in lymphoid blast crisis or Ph + ALL responded with 11/20 (55%) clearing their marrow blasts to 5%. Unfortunately, all but one of the lymphoid phenotype patients has relapsed between days 42 and 123. However, 7/38 (18%) of the myeloid blast crisis patients continue on STI571, in remission, with follow-up ranging from 101 to 349 days. The success of the phase I study prompted phase II studies; single-agent STI571 was tested further in interferon-refractory and interferon-intolerant patients, as well as accelerated-phase patients and patients with CML in myeloid blast crisis and Ph + ALL. These studies accrued over 1,000 patients at 27 centers in 6 countries over a period of 6-9 months; interim results from these studies were presented at the American Society of Hematology meeting in December of 2000 [9-11]. The phase II study included 532 chronic phase patients who were refractory to or intolerant of interferon, who were treated with 400 mg of STI571 daily. After a median exposure to STI571 of 254 days, with 86% of patients on therapy 6-12 months, 47% achieved major and 28% achieved complete cytogenetic responses. Only 3% of patients discontinued treatment due to disease progression with only 2% of patients stopping therapy due to adverse events. In the phase II study of the accelerated phase of CML [10], 233 patients were treated with either 400 or 600 mg of STI571. With similar follow-up to the chronic phase trial, 91% of patients showed a hematological response; 63% of patients achieved a complete hematological response but not all patients had recovery of peripheral blood counts. A complete hematological response, as well as peripheral blood recovery, was achieved by 44% of patients. Of note, 41% of patients on this trial had a cytogenetic response: 14% complete responses, 7% partial and 20% minor or minimal. Over

4 236 STI571: Targeting BCR-ABL as Therapy for CML 65% of patients were free of progression at 1 year and overall survival was 74% at 1 year. The results of the phase II study in the myeloid blast crisis of CML confirmed the response rate in the phase I clinical trials of STI571 [11]. Two hundred and sixty myeloid blast crisis patients received either 400 mg or 600 mg STI571. The overall response rate in this trial was 64%, with 11% of patients achieving a complete remission ( 5% marrow blasts) with peripheral blood recovery. Another 15% of patients cleared their marrows to less than 5% blasts but did not meet criteria for complete remission due to persistent cytopenias. Additionally, 38% of patients were either returned to chronic phase or had partial responses. Cytogenetic responses were seen in 27% of cases, with 15% major and 6% complete responses. Median survival was 6.8 months: 8.6 months in patients treated with STI571 as first-line therapy versus 4.4 months when STI571 was used as second-line therapy. Thirty percent of patients were still alive at 14 months, with a suggestion of a plateau on the survival curve. These results compare favorably in historical comparison to chemotherapy for myeloid blast crisis, which offers a median survival of approximately 3 months. Toxicity in all the STI571 trials has been low. The most common frequent side effects seen to date have been mild to moderate nausea, diarrhea, myalgias, and periorbital edema; less common side effects include skin rashes and peripheral edema. One fatal case of liver toxicity was seen; however, this was in the setting of concomitant significant acetaminophen administration. Myelosuppression has been seen, mainly in the accelerated and blast crisis trials, where 10%- 20% of patients experienced grade 3/4 cytopenias. This myelosuppression may be consistent with a therapeutic effect as the Ph + clone contributes the majority of hematopoiesis in these patients. In addition to the phase II clinical trials with STI571, a phase III trial randomizing newly diagnosed patients to either interferon with low-dose s.c. Ara-C versus STI571 is ongoing; this trial accrued rapidly and data collection is ongoing. LESSONS LEARNED AND FUTURE QUESTIONS The results with STI571 confirm the paramount importance of the deregulated tyrosine kinase activity of bcr-abl in the pathogenesis of CML. Integration of STI571 into CML treatment algorithms will require long-term follow-up data from the ongoing phase II and III clinical studies. Early in the chronic phase of CML, it is likely that bcr-abl is the sole oncogenic abnormality. Thus it is possible that STI571 could eradicate the malignant clone in a subset of patients. However, it is likely that this subset will be quite small and that combinations of STI571 with other agents will be required to achieve maximal therapeutic benefits in most patients. Prospectively identifying therapy with minimal toxicity and maximal efficacy for each patient is one of the goals of our current research. As more and more patients achieve cytogenetic responses through the availability of improved therapies, more sensitive assays such as real-time polymerase chain reaction will allow for better assessments of minimal residual disease and facilitate early relapse detection. Results observed in patients treated in accelerated phase and blast crisis of CML support the concept of partial dependence of the malignant clone(s) on bcr-abl tyrosine kinase activity with the probable acquisition of further molecular abnormalities resulting in escape and resistant disease. Several reports have described resistance mediated by bcrabl gene amplification in CML cell lines [12-14]; overexpression of multidrug resistance p-glycoprotein leading to drug efflux may be another mechanism. Evaluating patients who relapsed while on STI571, Gorre et al. [15] showed that the bcr-abl kinase activity was initially inhibited by STI571, but was reactivated in 18/18 patients at relapse. In 2/18 patients, this appeared to be due to bcr-abl amplification. Ex vivo, these cells remained sensitive to STI571 though requiring much higher doses of the inhibitor. These studies suggest that the mechanism of relapse in CML patients is dependent on a property intrinsic to the CML cells, such as bcr-abl amplification, drug efflux, or kinase domain mutation rendering the kinase insensitive to STI571 as opposed to proposed mechanisms such as protein binding [16]. As efforts continue to elucidate molecular mechanisms of resistance, current clinical trial efforts will focus on expanding the potential of STI571 by its use in combination therapy regimens. Examples of planned phase I/II trials include escalating high-dose Ara-C following initial therapy with STI571 in myeloid blast crisis and escalating doses of daunorubicin in combination with vincristine and prednisone following STI571 induction for lymphoid blast crisis. These studies are based on in vitro data [17] showing additive effects of daunorubicin and synergistic effects of Ara-C when combined with STI571. The in vitro studies also showed additive benefits when STI571 was combined with interferon. Thus new phase I/II trials in chronic phase patients with these combination therapies (STI571 + interferon and STI571 + lowdose Ara-C) will be initiated with the goal of achieving enhanced cytogenetic response (versus single-agent STI571) that could ultimately result in improved survival. The introduction of STI571 has supported further development of the model of CML pathogenesis and led to a new paradigm for CML therapy. Rational development of a potent and specific inhibitor of a target essential for disease pathogenesis has yielded high response rates and carries potential for the eradication of malignant clone.

5 Mauro, Druker 237 OTHER POTENTIAL TARGETS FOR STI571 Based on its ability to inhibit other tyrosine kinases, the spectrum of diseases that may respond to STI571 is growing. Gastrointestinal stromal tumors (GIST) are derived from the interstitial cells of Cajal which express the c-kit proto-oncogene [18]. The majority of GIST tumors have gain-of function mutations in c-kit with alterations in the extracellular, juxtamembrane, or kinase domains [19, 20]. Based on identification of c-kit mutations in GIST and the ability of STI571 to inhibit c-kit, clinical trials for patients with unresectable GIST were begun. STI571 has shown preliminary remarkable activity and clinical responses in patients with GIST [21-23]. There is a potential role for STI571 in other tumors bearing c-kit abnormalities, such as small cell lung cancer. Additionally, through its ability to inhibit the PGDFα receptor tyrosine kinase, STI571 may prove beneficial in chronic myelomonocytic leukemia based on its association with the (5;12) translocation and Tel-PDGFR fusion product. Finally, glioblastomas, the most common brain tumor and highly resistant to chemotherapy and radiation, are associated with an autocrine growth loop involving PDGF REFERENCES 1 Druker BJ, Lydon NB. Lessons learned from the development of an abl tyrosine kinase inhibitor for chronic myelogenous leukemia. J Clin Invest 2000;105: Yaish P, Gazit A, Gilon C et al. Blocking of EGF-dependent cell proliferation by EGF receptor kinase inhibitors. Science 1988;242: Druker BJ, Tamura S, Buchdunger E et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med 1996;2: le Coutre P, Mologni L, Cleris L et al. In vivo eradication of human BCR/ABL-positive leukemia cells with an ABL kinase inhibitor. 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Phase II study of STI571, a tyrosine kinase inhibitor, in patients (pts) with and its receptor. STI571 has been shown to inhibit the growth of glioblastoma cells injected into the brains of nude mice [24] suggesting that this agent could have potential as therapy for this currently incurable disease. CONCLUSIONS The introduction of STI571 has supported the model of CML pathogenesis and led to a new paradigm for CML therapy. Rational development of a potent and specific inhibitor of a target essential for disease pathogenesis has yielded high response rates and carries potential for the eradication of malignant clone. STI571 has already altered the therapeutic approach to CML and uses in other malignancies will be sought. Recent advances in both target and drug discovery should greatly accelerate the pace of generating drugs such as STI571 for other cancers and leukemias. ACKNOWLEDGMENT B.J.D. serves as a consultant to Novartis Pharmaceuticals. resistant or refractory Philadelphia chromosome-positive chronic myelogenous leukemia (Ph + CML). Blood 2000;96:470a. 10 Talpaz M, Silver RT, Druker B et al. A phase II study of STI571 in adult patients with Philadelphia chromosome positive chronic myeloid leukemia in accelerated phase. Blood 2000;96:470a. 11 Sawyers C, Hochhaus A, Feldman E et al. A phase II study to determine the saftey and anti-leukemic effects of STI571 in patients with Philadelphia chromosome positive chronic myeloid leukemia in myeloid blast crisis. Blood 2000;96:470a. 12 le Coutre P, Tassi E, Varella-Garcia M et al. Induction of resistance to the Abelson inhibitor STI571 in human leukemia cells through gene amplification. Blood 2000;95: Weisberg E, Griffin JD. Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL-transformed hematopoietic cell lines. Blood 2000;95: Mahon FX, Deininger MW, Schultheis B et al. 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6 238 STI571: Targeting BCR-ABL as Therapy for CML 17 Thiesing JT, Ohno-Jones S, Kolibaba KS et al. Efficacy of STI571, an ABL tyrosine kinase inhibitor in conjunction with other anti-neoplastic agents against BCR-ABL positive cells. Blood 2000;96: Kindblom LG, Remotti HE, Aldenborg F et al. Gastrointestinal pacemaker cell tumor (GIPACT): gastrointestinal stromal tumors show phenotypic characteristics of the interstitial cells of Cajal. Am J Pathol 1998;152: Hirota S, Isozaki K, Moriyama Y et al. Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors. Science 1998;279: Lux ML, Rubin BP, Biase TL et al. KIT extracellular and kinase domain mutations in gastrointestinal stromal tumors. Am J Pathol 2000;156: Joensuu H, Roberts PJ, Sarlomo-Rikala M et al. Effects of tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N Engl J Med 2001;344: Van Oosterom AT, Judson I, Verweij J et al. STI571, an active drug in metastatic gastro intestinal stromal tumors (GIST), an EORTC phase I study. proc Am Soc Clin Oncol 2001;20:1a. 23 Blanke CD, von Mehren M, Joensuu H et al. Evaluation of the safety and efficacy of an oral molecularly-targeted therapy, STI571, in patients with unresectable or metastatic gastrointestinal stromal tumors (GISTS) expressing c-kit (CD117). Proc Am Soc Clin Oncol 2001:20:1a. 24 Alberta JA, Zdunek PR, Acar M et al. Intracranial inhibition of platelet-derived growth factor-mediated glioblastoma cell growth by an orally active kinase inhibitor of the 2-phenylaminopyrimidine class. Cancer Res 2000;60: