Tyrosine Kinase Inhibitors: Targeting Considerations

Michael C. Heinrich; Charles D. Blanke; Christopher L. Corless; Brian J. Druker

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Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003.

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Holland-Frei Cancer Medicine. 6th edition.

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Tyrosine Kinase Inhibitors: Targeting Considerations

Michael C. Heinrich, MD, Charles D. Blanke, MD, Christopher L. Corless, MD, PhD, and Brian J. Druker, MD.

Whether a cancer can be treated effectively with a tyrosine kinase inhibitor (TKI) may depend largely on the extent to which the growth of the tumor cells is dependent on the targeted tyrosine kinase(s). This principle can be illustrated by hypothetical tumor cells whose growth is dependent upon tyrosine kinase A, kinase B, or both kinases A + B (Figure 54-1). The growth of tumor cells that are dependent upon a single kinase would be inhibited by treatment with the appropriate TKI. In contrast, there would be no effect on proliferation when cells dependent upon kinase A are treated with an inhibitor against kinase B, or vice versa. In the case of tumor cells that are dependent upon both kinases A + B, therapy with an inhibitor against one of the kinases would reduce proliferation by only 50%. Complete inhibition of such cells could be achieved only when both kinases are inhibited. This model is based on actual experimental observations with factor-dependent human acute myelogenous leukemia (AML) cells.¹⁸ In some cancers, the situation may be even more complex, with tumor cell survival dependent upon three or more kinases. In such cases, it could be necessary to target all of the kinases, or to inhibit downstream effectors that were shared between all or most of the kinases.

Figure 54-1

Effect of tyrosine kinase inhibitors (TKIs) against kinase A or kinase B in tumors dependent upon A and/or B for proliferation. The clinical effect of monotherapy with a given TKI is determined by the dependence of the tumor cells upon the targets inhibited (more...)

The implications of this model are that monotherapy with a TKI will be most effective when the growth of a tumor is dependent on only one tyrosine kinase. As described in this chapter, chronic myelogenous leukemia (CML) and gastrointestinal stromal tumor (GIST) both fit this description. Whether this will be the case with other tumors is unknown. It is likely that early in the development of a tumor, single genetic defects are more common, while in advanced malignancies, multiple, redundant pathways support the growth and survival of the tumor. For this reason, monotherapy with a TKI may be most useful in the earlier stages disease, if activation of a specific tyrosine kinase is critical to the development of the tumor.¹⁹ In this regard, the most attractive cancers for monotherapy with a TKI are those diseases in which there is a genomic mechanism of target activation (see Table 54-1).

In the above model it should be noted that a biologic effect does not necessarily equate with a clinical benefit. For example, in the case of a tumor that is codependent upon kinase A and B, treatment with an inhibitor specific to kinase A would decrease proliferation by 50% (a biologic effect). However, because slowing the growth rate would not decrease the tumor size, this result would be regarded as “progressive disease.” If the end point of the clinical trial is an objective response rate, the inhibitor would be dismissed as an ineffective therapy. However, end points such as delay in time to progression or prolongation of survival might reveal a therapeutic benefit. Additional studies that combined the kinase A inhibitor with other treatments (kinase B inhibitors, cytotoxics, immune treatments, etc) that could impact the other 50% of proliferation might result in improved clinical efficacy.

These issues demonstrate some of the difficulties faced in the development of targeted agents such as TKIs. Specifically, single-agent TKIs may have minimal activity using standard endpoints in patients with advanced malignancies and there may be a need to combine TKIs with other agents to see significant clinical activity. Besides the need to consider alternative endpoints, it is also crucial to include measurement of kinase activation before and during therapy. For example, if the targeted kinase is not inhibited in the tumor, this raises the possibility of poor tumor penetration or lack of potency of the agent. If, however, the kinase is inhibited and no measurable response is observed, this suggests that either the kinase is not required for the growth and survival of the cancer and that an agent targeting this kinase will not be clinically useful, or that an incorrect end point was selected to determine response. As is discussed later, it is also possible that only a subset of patients whose cancer expresses a target will respond to therapy. Thus, clinical studies of kinase inhibitors will need to integrate sophisticated correlative studies with various clinical end points to draw meaningful conclusions about monotherapy and to guide development of combination therapy studies.

BCR-ABL as a Therapeutic Target

Of all the diseases associated with activated tyrosine kinases, the best-validated target is the BCR-ABL tyrosine kinase in CML. The pathogenetic role of this kinase in CML was firmly established by a series of discoveries over the last 40 years. Working in Philadelphia, Nowell and Hungerford recognized the association of a shortened chromosome with CML.²⁰ This chromosome, later recognized to be a shortened chromosome 22 and referred to as the Philadelphia chromosome (Ph), results from a reciprocal translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;q11).²¹ This translocation results in fusion of the ABL oncogene from chromosome 9 with sequences from chromosome 22, the breakpoint cluster region (BCR), giving rise to a chimeric BCR-ABL gene.^22,²³ This fusion gene is transcribed and translated into a protein that functions as a constitutively activated tyrosine kinase.^24,²⁵ Depending on the site of the breakpoint in bcr, one of two different fusion proteins are produced, p185 (185 kilodaltons [kDa]) and p210 (210 kDa). The p210 protein is present in approximately 95% of patients with CML and in up to 20% of adult patients with de novo acute lymphoblastic leukemia (ALL), whereas the p185 form is seen in approximately 10% of adult patients with ALL and in the majority of pediatric patients with Ph-positive ALL (5% of pediatric ALL cases).^26,²⁷

Expression of BCR-ABL in a variety of myeloid and lymphoid cell lines renders these cell lines growth factor independent for proliferation.^28–30 Similar results have been obtained using primary hematopoietic cells.^31,³² However, even more convincing evidence for the leukemogenic potential of BCR-ABL has been demonstrated in animal models. In one set of experiments, transgenic mice that expressed BCR-ABL were shown to develop a rapidly fatal acute leukemia.³³ Using a different approach, a BCR-ABL-expressing retrovirus was used to infect murine bone marrow. These BCR-ABL-expressing marrow cells were used to repopulate irradiated mice. The transplanted mice developed a variety of myeloproliferative disorders, including a CML syndrome.^34,³⁵ Although these models demonstrated the leukemogenic potential of BCR-ABL, it is possible that secondary changes were required for leukemia to develop. Recently, Huettner and colleagues placed BCR-ABL under the control of a tetracycline repressible promoter.³⁶ Mice expressing this transgene developed a reversible leukemia dependent on the presence or absence of tetracycline, clearly demonstrating the leukemic potential of BCR-ABL acting as the sole oncogenic abnormality. As all of the transforming activities of BCR-ABL are dependent on its tyrosine kinase activity,³⁷ an inhibitor of the BCR-ABL kinase would be predicted to be an effective and selective therapeutic agent for CML.

Preclinical Development of Imatinib

Compounds that possess inhibitory activity for protein tyrosine kinases were initially isolated from natural sources in the early to mid 1980s. These compounds include the flavanoid quercetin, the isoflavonoid genistein, the antibiotic herbimycin A, and erbstatin.^38–40 Although these compounds can revert cells transformed by tyrosine kinase oncogenes to a nontransformed phenotype, none of these compounds have demonstrated specificity among tyrosine kinases. In the late 1980s, Yaish and colleagues synthesized a series of compounds, known as tyrphostins, that were the first compounds to display specificity among tyrosine kinases.⁸ One of the tyrphostins, AG1112, inhibits the ABL protein tyrosine kinase, and induces differentiation and death of a BCR-ABL-positive, erythroid blast crisis, CML cell line, K562.^41,⁴² Similar data for induction of K562 differentiation have been obtained with genistein, herbimycin A, and erbstatin.^43–45

Working independently, scientists at Ciba-Geigy (now Novartis Pharma) identified a 2-phenylaminopyrimidine as a weakly potent and nonspecific kinase inhibitor. A series of related compounds were then synthesized and using structure-activity relationships, these compounds were optimized against a variety of targets.^17,⁴⁶ STI571 (signal-transduction inhibitor) was one of many compounds developed in this program and was found to be a potent inhibitor of the platelet-derived growth factor receptor (PDGFR) and the ABL tyrosine kinases. Further testing revealed that it was relatively selective for the ABL tyrosine kinases, including BCR-ABL, ABL, v-ABL, and ARG (Abelson-related gene).^46,⁴⁷ Besides the ABL and PDGFR α and β tyrosine kinases, the only other tyrosine kinase inhibited by STI571 is KIT.¹⁷ STI571 (formerly CGP57148, now Glivec, Gleevec, or imatinib) emerged as the lead compound for clinical development based on its superior in vitro selectivity against CML cells and its drug-like properties, including pharmacokinetic and formulation properties.

Initial studies of imatinib showed that it specifically inhibited the proliferation of BCR-ABL-expressing cells.⁴⁸ These studies have been confirmed by numerous laboratories and demonstrate that imatinib, at concentrations of 1 and 10 μM, kills or inhibits the proliferation of all BCR-ABL-expressing cell lines tested to date.^46,^49–52 In contrast, a variety of immortalized or transformed cell lines that do not express BCR-ABL are not sensitive to imatinib. Studies in mice also showed that imatinib had in vivo activity against BCR-ABL-expressing cells. Initial experiments failed to eradicate BCR-ABL-expressing tumors.⁴⁸ However, subsequent experiments that used a three-times-per-day administration schedule that allowed continuous exposure to imatinib eradicated the tumor cells.⁵³ This suggested that continuous exposure to imatinib would be important for optimal antileukemic effects in human clinical trials.

Clinical Studies of Imatinib in CML

A standard dose-escalation, Phase I study of imatinib was initiated in June 1998, and results of this study were published.^19,⁵⁴ Imatinib was well tolerated with minimal side effects. Despite dose escalation from 25 mg to 1,000 mg in 14 cohorts of patients, a maximally tolerated dose could not be defined. Imatinib was administered once daily and pharmacokinetics showed a half-life of 13 to 16 hours. Significant clinical benefits were observed at doses of 300 mgand above. In chronic phase patients who had failed therapy with interferon, 53 of 54 (98%) achieved a complete hematologic response and 96% of these responses lasted beyond 1 year. In myeloid blast crisis patients, 21 of 38 (55%) patients responded, with 18% having responses lasting beyond 1 year.

The success of the Phase I studies prompted Phase II studies in which single-agent imatinib 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 Philadelphia chromosome-positive ALL. These studies accrued more than 1,000 patients at 30 centers in 6 countries over a period of 6 to 9 months. Figure 54-2 summarizes the published results from these studies with 18 months of follow-up.^55–58 These studies served as the basis for accelerated FDA approval of imatinib in May 2001 for the above indications.

Figure 54-2

Phase II and Phase III results of imatinib for CML. The results shown are for newly diagnosed chronic-phase patients with a median follow-up of 14 months. For the Phase II studies in chronic-phase patients who failed interferon therapy, accelerated-phase (more...)

There were 532 chronic-phase patients who were refractory to, or intolerant of, interferon-α who were treated with an imatinib dose of 400 mg daily; 95% of these patients achieved a complete hematologic response (CHR), and with a median follow-up of 18 months, the estimated progression-free survival was 89%. Only 2% of patients discontinued therapy because of adverse events.⁵⁸ Of 235 accelerated-phase patients treated with 400 or 600 mg of imatinib daily, 82% showed some form of hematologic response, with 34% achieving a CHR. Estimated 12-month progression-free and overall survival rates were 59% and 74%, respectively. Patients treated with 600 mg had trends towards higher hematologic and cytogenetic response rates, with statistically significant improvements in time to disease progression and overall survival.⁵⁷ Results of the Phase II study treating 260 myeloid blast-crisis patients with imatinib showed an overall response rate of 52%, with 8% achieving complete remission (CR = < 5% blasts) with peripheral blood recovery.⁵⁶ Another 4% of patients cleared their marrows to less than 5% blasts but did not meet the criteria for CR because of persistent cytopenias. Lastly, 18% of patients were either returned to chronic phase or had partial responses. Median survival was 6.9 months; 20% of patients were still alive at 18 months, with a suggestion of a plateau on the survival curve. These results compare favorably to historical controls treated with chemotherapy for myeloid blast crisis in which the median survival is approximately 3 months.⁵⁶ In patients with Ph-positive acute lymphoblastic leukemia, 29 of 48 patients (60%) responded to single-agent imatinib. However, the duration of response was relatively short with a median estimated time-to-disease progression of only 2.2 months.⁵⁵

A Phase III randomized study comparing imatinib to interferon plus cytarabine in newly diagnosed patients accrued more than 1,000 patients in a 6-month period and data collection is ongoing. Preliminary results presented at the American Society of Clinical Oncology meeting in May 2002 showed that with a median follow-up of 14 months, patients randomized to imatinib had statistically significant better results than patients treated with interferon plus cytarabine in all parameters measured, including rates of complete hematologic response, major and complete cytogenetic response, tolerance of therapy, and freedom from disease progression.⁵⁹

Mechanisms of Relapse/Resistance to Imatinib

Response rates to imatinib in chronic-phase patients are quite high and thus far, responses have been durable. Response rates are also quite high in patients with advanced-phase disease, but relapses, despite continued therapy with imatinib, are common. An analysis of the expression of the target should be the starting point for evaluation of relapse mechanisms (Figure 54-3), and in all patients who have relapsed, the BCR-ABL kinase remains present. A particularly useful categorization of relapsed/resistant CML patients has been to determine whether or not there is persistent inhibition of the BCR-ABL kinase (Figure 54-3). Patients with persistent inhibition of the BCR-ABL kinase would be predicted to have additional molecular abnormalities besides BCR-ABL driving the growth and survival of the malignant clone. In contrast, patients with reactivation of the kinase would be postulated to have resistance mechanisms that either prevent imatinib from reaching the target or render the target insensitive to BCR-ABL. In the former category are mechanisms such as drug efflux or protein binding of imatinib. In the latter category would be mutations of the BCR-ABL kinase that render BCR-ABL insensitive to imatinib and amplification of the BCR-ABL protein.

Figure 54-3

Distinguishing between potential mechanisms of relapse.

In the largest studies of resistance or relapse, several consistent themes have emerged. In the majority of patients who respond to imatinib but subsequently relapse while remaining on therapy, the BCR-ABL kinase has been reactivated.⁶⁰ BCR-ABL kinase activity was analyzed by assessing tyrosine phosphorylation of CRKL, a direct substrate of the BCR-ABL kinase, and the major tyrosine phosphorylated protein in CML patient samples.^60,⁶¹ In these studies, more than 50%, and perhaps as many as 90%, of patients with hematologic relapse have BCR-ABL point mutations in at least 13 different amino acids scattered throughout the Abl kinase domain (Figure 54-4).^62–67 Some other patients have amplification of BCR-ABL at the genomic or transcript level. In contrast, in patients with primary resistance, that is, patients who do not respond to imatinib therapy, BCR-ABL-independent mechanisms are most common.⁶⁷

Figure 54-4

Schematic of point mutations in the Abl kinase domain. The Abl kinase domain, from amino acid 240 to 500, is shown with the ATP binding domain (P), the catalytic domain (C), and the activation loop (A). The numbers below the kinase domain are amino acids (more...)

In patients who relapse as a consequence of reactivation of the BCR-ABL kinase, the BCR-ABL kinase remains a good target. Analysis of the inhibitory activity of imatinib against these mutations shows that some mutations might be sensitive to dose escalation, but the most common mutation at amino acid 315 is completely insensitive to imatinib.⁶⁸ ABL kinase inhibitors with specificity that differs from imatinib have already been synthesized.^69–71 One of these compounds, PD180970, is capable of inhibiting some, but not all of the common BCR-ABL kinase mutations.⁷² These data suggest that it may be possible to treat patients with several different ABL kinase inhibitors to circumvent resistance. Given that BCR-ABL kinase activity has been reactivated in relapsed patients, it might also be useful to target signaling pathways activated by BCR-ABL, such as RAF/MEK/ERK, PI-3 kinase, AKT, or RAS. For example, two groups recently reported in vitro sensitivity of imatinib resistant BCR-ABL-positive cell lines to a farnesyl transferase inhibitor.^73,⁷⁴ Moreover, Hoover and colleagues, observed that this compound sensitized cells to imatinib, even imatinib-resistant cell lines.⁷³ Alternatively, strategies to increase BCR-ABL protein degradation by using agents such as geldanamycin, 17-AAG, or arsenic trioxide might be useful.^74–76

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Heinrich MC, Blanke CD, Corless CL, et al. Tyrosine Kinase Inhibitors: Targeting Considerations. In: Kufe DW, Pollock RE, Weichselbaum RR, et al., editors. Holland-Frei Cancer Medicine. 6th edition. Hamilton (ON): BC Decker; 2003.