Targeted Therapy with Small Molecule Kinase Inhibitors
Charles L. Sawyers
In 2001 the first tyrosine kinase inhibitor, imatinib, was approved for clinical use in chronic myeloid leukemia (CML). The spectacular success of this first-in-class agent ushered in a transformation in cancer drug discovery from efforts that were largely based on novel cytotoxic chemotherapy agents to an almost exclusive focus on molecularly targeted agents across the pharmaceutical and biotechnology industry and academia. This chapter summarizes this remarkable decade of progress with the focus on the concepts underlying this paradigm shift as well as the considerable challenges that remain (Tables 13.1 and 13.2). Readers in search of more specific details on individual drugs and their indications should consult the relevant disease-specific chapters elsewhere in this volume as well as references cited within this chapter. Readers should also note that the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinases covered here have also been successfully targeted by monoclonal antibodies that engage these proteins at the cell surface. These drugs, often referred to as biologics rather than small molecule inhibitors, are covered in other chapters. The chapter is organized around kinase targets rather than diseases and, intentionally, has a historical flow to make certain thematic points and illustrate the broad lessons that have been and continue to be learned through the clinical development of these exciting agents.
Perhaps the most stunning discovery from the clinical trials of the ABL kinase inhibitor imatinib was the recognition that tumor cells acquire exquisite dependence on the BCR-ABL fusion oncogene, created by the Philadelphia chromosome translocation.1 Although this may seem intuitive at first glance, consider the fact that the translocation arises in an otherwise normal hematopoietic stem cell whose survival is regulated by a complex array of growth factors and interactions with the bone marrow microenvironment. While BCR-ABL clearly gives this cell a growth advantage that, over years, results in the clinical phenotype of CML, there was no reason to expect that these cells would depend on BCR-ABL for their survival when confronted with an inhibitor. In the absence of BCR-ABL, these tumor cells could presumably rely on the marrow microenvironment for their survival, just like their normal nontransformed neighbors. Thus, it seemed more likely that by shutting down the driver oncogene, BCR-ABL inhibitors would halt the progression of CML but not eliminate the pre-existing tumor cells. But in fact, CML progenitors are eliminated after just a few months of anti-BCR-ABL therapy, indicating they are dependent on the driver oncogene for their survival and have “forgotten” how to return to normal. This phenomenon, subsequently documented in a variety of human malignancies, is colloquially termed “oncogene addiction.”2 Although the molecular basis for this addiction remains to be defined, the notion of finding an Achilles’ heel for each cancer has captivated the cancer research community, spawning a broad array of efforts to elucidate the molecular identity of these targets and discover relevant inhibitors.
EARLY SUCCESSES: TARGETING CANCERS WITH WELL-KNOWN KINASE MUTATIONS
From the beginning, clinical trials of imatinib were restricted to patients with Philadelphia chromosome -positive CML. For what seem like obvious reasons, there was never any serious discussion about treating patients with Philadelphia chromosome -negative leukemia because the assumption was that only patients with the BCR-ABL fusion gene would have a chance of responding. This was clearly a wise decision because hematologic response rates approached 90% and cytogenetic remissions were seen in nearly half of the patient in the early phase studies.3 It was obvious the drug worked and imatinib was approved in record time. Unwittingly, the power of genome-based patient selection was demonstrated in the clinical development of the very first kinase inhibitor. It has
taken a decade for this lesson to be fully learned. Today the much larger clinical experience with an array of different kinase inhibitors across many tumor types has led to a much better understanding of the principles that dictate oncogene addiction that, in retrospect, were staring researchers in the face. Foremost among them is the notion that tumors with somatic mutation or amplification of a kinase drug target are much more likely to be dependent on that target for survival. Hence, a patient whose tumor has such a mutation is much more likely to respond to treatment with the appropriate inhibitor.
taken a decade for this lesson to be fully learned. Today the much larger clinical experience with an array of different kinase inhibitors across many tumor types has led to a much better understanding of the principles that dictate oncogene addiction that, in retrospect, were staring researchers in the face. Foremost among them is the notion that tumors with somatic mutation or amplification of a kinase drug target are much more likely to be dependent on that target for survival. Hence, a patient whose tumor has such a mutation is much more likely to respond to treatment with the appropriate inhibitor.
TABLE 13.1 APPROVED KINASE INHIBITORS | |||||||||||||||||||||||||||||||
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After CML, the next example to illustrate this principle was gastrointestinal stromal tumor (GIST), which is associated with mutations in the KIT tyrosine kinase receptor or, more rarely, in the platelet-derived growth factor receptor (PDGFR).4,5 Serendipitously, imatinib inhibits both KIT and PDGFR; therefore, the clinical test of KIT inhibition in GIST followed quickly on the heels of the success in CML.6 In retrospect, the rapid progress made in these two diseases was based, in part, on the fact that the driver molecular lesion (BCR-ABL or KIT mutation, respectively) is present in nearly all patients who are diagnosed with these two diseases. The molecular analysis merely confirmed the diagnosis that was made using standard clinical and histological criteria. Consequently, clinicians could identify the patients most likely to respond based on clinical criteria rather than rely on an elaborate molecular profiling infrastructure to prescreen patients. Consequently, clinical trials evaluating kinase inhibitors in CML and GIST accrued quickly and the therapeutic benefit became clear almost immediately.
The notion that molecular alteration of a driver kinase determines sensitivity to a cognate kinase inhibitor was further validated during the development of the dual EGFR/HER2 kinase inhibitor lapatinib. Clinical trials of this kinase inhibitor were conducted in women with advanced HER2-positive breast cancer based on earlier success in these same patients with the monoclonal antibody trastuzumab that targets the extracellular domain of the HER2 kinase. Lapatinib was initially approved in combination with the cytotoxic agent capecitabine for women with resistance to
trastuzumab,7 then was subsequently approved for frontline use in metastatic breast cancer in combination with chemotherapy or hormonal therapy, depending on estrogen-receptor status. A key ingredient that enabled the clinical development of lapatinib was the routine use of HER2 gene amplification testing in the diagnosis of breast cancer, pioneered during the development of trastuzumab several years earlier. This widespread clinical practice allowed rapid identification of those patients most likely to benefit. If lapatinib trials had been conducted in unselected patients, the clinical signal in breast cancer would likely have been missed.
trastuzumab,7 then was subsequently approved for frontline use in metastatic breast cancer in combination with chemotherapy or hormonal therapy, depending on estrogen-receptor status. A key ingredient that enabled the clinical development of lapatinib was the routine use of HER2 gene amplification testing in the diagnosis of breast cancer, pioneered during the development of trastuzumab several years earlier. This widespread clinical practice allowed rapid identification of those patients most likely to benefit. If lapatinib trials had been conducted in unselected patients, the clinical signal in breast cancer would likely have been missed.
TABLE 13.2 UNAPPROVED KINASE INHIBITORS WITH CLEAR EVIDENCE OF CLINICAL ACTIVITY | |||||||||||||||||||||||||||||||||||||||||||||
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The Serendipity of Unexpected Clinical Responses
In contrast to the logical development of imatinib and lapatinib in molecularly defined patient populations, the EGFR kinase inhibitors gefitinib and erlotinib entered the clinic without the benefit of such a focused clinical development plan. Although considerable preclinical data implicated EGFR as a cancer drug target, there was little insight into which patients were most likely to benefit. The first clue that EGFR inhibitors would have a role in lung cancer came from the recognition, by several astute clinicians, of remarkable responses in a small fraction of patients with lung adenocarcinoma.8 Further studies revealed the curious clinical circumstance that those patients most likely to benefit tended to be never smokers, women, and those of Asian ethnicity.9 Clearly there was a strong clinical signal in a subgroup of patients who could perhaps be enriched based on these clinical features, but it seemed that a unifying molecular lesion must be present. Three academic groups simultaneously converged on the answer. Mutations in the EGFR gene were detected in the 10% to 15% of patients
with lung adenocarcinoma who had radiographic responses.10,11,12 It may seem surprising that mutations in a gene as highly visible as EGFR and in such a prevalent cancer had not been detected earlier. But the motivation to search aggressively for EGFR mutations was not there until the clinical responses were seen. Perhaps even more surprising was the failure of the pharmaceutical company sponsors of the two most advanced compounds, gefitinib and erlotinib, to embrace this important discovery and refocus future clinical development plans on patients with EGFR mutant lung adenocarcinoma.
with lung adenocarcinoma who had radiographic responses.10,11,12 It may seem surprising that mutations in a gene as highly visible as EGFR and in such a prevalent cancer had not been detected earlier. But the motivation to search aggressively for EGFR mutations was not there until the clinical responses were seen. Perhaps even more surprising was the failure of the pharmaceutical company sponsors of the two most advanced compounds, gefitinib and erlotinib, to embrace this important discovery and refocus future clinical development plans on patients with EGFR mutant lung adenocarcinoma.
Clinical development of cytotoxic agents has always proceeded empirically. Typically, small numbers of patients with different cancers are treated in “all comer” phase 1 studies (no enrichment for subgroups) with the goal of eliciting a clinical signal in at least one tumor type. A single agent response rate of 20% to 30% in a diseasespecific phase 2 trial can justify a randomized phase 3 registration trial where the typical end point for drug approval is time to progression or survival. Cytotoxics are also typically evaluated in combination with existing standard of care treatment (typically approved chemotherapy agents) with the goal of increasing the response rate or enhancing the duration of response.
The clinical development of gefitinib and erlotinib followed the cytotoxic model. Both drugs had similarly low but convincing single agent response rates (10% to 15%) in chemotherapyrefractory advanced lung cancer. Indeed, gefitinib was originally granted accelerated approval by the U.S. Food and Drug Administration (FDA) in 2003 based on the impressive nature of these responses, contingent on the completion of formal phase 3 studies with survival end points.13 The sponsors of both drugs therefore conducted phase 3 registration studies in patients with chemotherapyrefractory advanced stage lung cancer but without prescreening patients for EGFR mutation status. (In fairness, these trials were initiated prior to the discovery of EGFR mutations in lung cancer.) Erlotinib was approved in 2004 on the basis of a modest survival advantage over placebo (the BR.21 trial); however, gefitinib failed to demonstrate a survival advantage in essentially the same patient population.14,15 This difference in outcome was surprising since the two drugs have highly similar chemical structures and biological properties. Perhaps the most important difference in the two trials was drug dose. Erlotinib was given at the maximum tolerated dose, which produces a high frequency of rash and diarrhea. Both side effects are presumed “on target” consequences of EGFR inhibition since EGFR is highly expressed in skin and gastrointestinal epithelial cells. In contrast, gefitinib was dosed slightly lower to mitigate these toxicities, with the rationale that responses should not be compromised since clinical responses were clearly documented at lower doses.
In parallel with the single agent phase 3 trials in chemotherapy-refractory patients, both gefitinib and erlotinib were studied as upfront therapy for advanced lung cancer to determine if either would improve the efficacy of standard “doublet” (carboplatin/paclitaxel or gemcitabine/cisplatin) chemotherapy when all three drugs were given in combination. These trials, termed INTACT-1 and INTACT-2 (gefitinib with either gemcitabine/cisplatin or with carboplatin/paclitaxel) and TRIBUTE (erlotinib with carboplatin/paclitaxel) collectively enrolled over 3,000 patients.16,17,18 Excitement in the oncology community was high based on the clear single agent activity of both EGFR inhibitors. But both trials were spectacular failures—neither drug showed any benefit over chemotherapy alone. The emerging data on EGFR mutations in the 10% to 15% of patients who responded to single agent EGFR inhibitor therapy provided a logical explanation. Only a small fraction of patients enrolled in these phase 3 trials had a chance of benefit since the clinical signal from those whose tumors had EGFR mutations was diluted by all the patients whose tumors had no EGFR alterations, many of whom benefited from chemotherapy.
The convergence of the EGFR mutation discovery with these clinical trial results will be remembered as a remarkable time in the history of targeted cancer therapies, not just for the important role of these agents as lung cancer therapies but for the delays in studying whether EGFR genotype should drive treatment selection. Perhaps the most egregious error came from a retrospective analysis of tumors from patients treated in the BR.21 trial that concluded that EGFR mutations did not predict for a survival advantage.19 (EGFR gene amplification was associated with survival but only in univariate analysis.) This conclusion was concerning because less than 30% of patients on the trial had tissue available for EGFR mutation analysis, raising questions about the adequacy of the sample size. Furthermore, the EGFR mutation assay used by the authors was subsequently criticized because a significant number of the EGFR mutations reported in these patients were in residues not previously found by others who had sequenced thousands of tumors. Many of these mutations were suspected to be an artifact of working from formalin-fixed biopsies. (Formalin fixed, paraffin-embedded tissue samples present special challenges for nucleic acid analysis that can be avoided if the tissue is fresh frozen at the time of acquisition or can be overcome with additional controls.)
More recently, clinical investigators in Asia, where a greater fraction of lung cancers (roughly 30%) are positive for EGFR mutations, addressed the question of whether mutations predict for clinical benefit in a prospective trial. In this study known as IPASS, gefitinib was clearly superior to standard doublet chemotherapy as frontline therapy for patients with advanced EGFR mutation-positive lung adenocarcinoma.20 Conversely, EGFR
mutation-negative patients fared much worse with gefitinib and benefited from chemotherapy. In addition, EGFR mutation-positive patients had a more favorable overall prognosis regardless of treatment, indicating that EGFR mutation is also a prognostic biomarker. The IPASS trial serves as a compelling example of a properly designed (and executed) biomarker-driven clinical trial. Although the rationale for this clinical development strategy had been demonstrated years earlier with BCR-ABL in leukemia, KIT in GIST, and HER2 in breast cancer, it was difficult to derail the empiric approach that had been used for decades in developing cytotoxic agents.
mutation-negative patients fared much worse with gefitinib and benefited from chemotherapy. In addition, EGFR mutation-positive patients had a more favorable overall prognosis regardless of treatment, indicating that EGFR mutation is also a prognostic biomarker. The IPASS trial serves as a compelling example of a properly designed (and executed) biomarker-driven clinical trial. Although the rationale for this clinical development strategy had been demonstrated years earlier with BCR-ABL in leukemia, KIT in GIST, and HER2 in breast cancer, it was difficult to derail the empiric approach that had been used for decades in developing cytotoxic agents.
Platelet-Driven Growth Factor Receptor Leukemias and Sarcoma
The discovery of EGFR mutations in lung cancer (motivated by dramatic clinical responses in a subset of patients treated with EGFR kinase inhibitors) is the most visible example of the power of bedside-to-bench science, but it is not the only (or the first) such example from the kinase inhibitor era. Shortly after the approval of imatinib for CML in 2001, two case reports documented dramatic remissions in patients with hypereosinophilic syndrome (HES), a blood disorder characterized by prolonged elevation of eosinophil counts and subsequent organ dysfunction from eosinophil infiltration, when treated with imatinib.21,22 Although HES resembles myeloproliferative diseases such as chronic myeloid leukemia, the molecular pathogenesis of HES was completely unknown at the time. Reasoning that these clinical responses must be explained by inhibition of a driver kinase, a team of laboratory-based physician scientists quickly searched for mutations in the three kinases known to be inhibited by imatinib (ABL, KIT, and PDGFR). ABL and KIT were quickly excluded, but the PDGFRα gene was targeted by an interstitial deletion that fused the upstream FIP1L1 gene to PDGFRα.23 FIP1L1-PDGFRα is a constitutively active tyrosine kinase, analogous to BCR-ABL, and also inhibited by imatinib. As with EGFR-mutant lung cancer, the molecular pathophysiology of HES was discovered by dissecting the mechanism of response to the drug used to treat it.
The HES/FIP1L1-PDGFRα story serves as a nice bookend to an earlier discovery that the t(5,12) chromosome translocation, found rarely in patients with chronic myelomonocytic leukemia, creates the TEL-PDGFRβ fusion tyrosine kinase.24 Similar to HES, treatment of patients with t(5,12) translocation-positive leukemias with imatinib has also proven successful.25 A third example comes from dermatofibrosarcoma protuberans, a sarcoma characterized by a t(17,22) translocation that fuses the COL1A gene to the gene for PDGFB ligand (not the receptor). COL1A-PDGFB is oncogenic through autocrine stimulation of the normal PDGFR in these tumor cells. Patients with dermatofibrosarcoma protuberans respond to imatinib therapy because it targets the PDGFR, just one step downstream from the oncogenic lesion.26
Exploiting the New Paradigm: Searching for Other Kinase-Driven Cancers
The benefits of serendipity notwithstanding, the growing number of examples of successful kinase inhibitor therapy in tumors with mutation or amplification of the drug target begged for a more rational approach to drug discovery and development. In 2002, the list of human tumors known to have mutations in kinases was quite small. Due to advances in automated gene sequencing, it became possible to ask whether a much larger fraction of human cancers might also have such mutations through a brute force approach. To address this question comprehensively, one would have to sequence all of the kinases in the genome in hundreds of samples of each tumor type. Several early pilot studies demonstrated the potential of this approach by revealing important new targets for drug development. Perhaps the most spectacular was the discovery of mutations in the BRAF kinase in over half of patients with melanoma, as well as in a smaller fraction of colon and thyroid cancers27 Another was the discovery of mutations in the Janus-associated tyrosine kinase 2 (JAK2) in nearly all patients with polycythemia vera, as well as a significant fraction of patients with myelofibrosis and essential thrombocytosis.28,29,30 A third example was the identification of PIK3CA mutations in a variety of tumors, with the greatest frequencies in breast, endometrial, and colorectal cancers.31 PIK3CA encodes a lipid kinase that generates the second messenger phosphatidyl inositol 3-phosphate (PIP3). PIP3 activates growth and survival signaling through the AKT family of kinases as well as other downstream effectors. Coupled with the well-established role of the PTEN (phosphatase and tensin homologue) lipid phosphatase in dephosphorylating PIP3, the discovery of PIK3CA mutations focused tremendous attention on developing inhibitors at multiple levels of this pathway, as discussed further below.
Each of these important discoveries—BRAF, JAK2, PIK3CA—came from relatively small efforts (less than 100 tumors) generally focused on resequencing only those exons that coded for regions of kinases where mutations had been found in other kinases (typically the juxtamembrane and kinase domains). These restricted searches were largely driven by cost. In 2006, a comprehensive effort to sequence all of the exons in all kinases in 100 tumors could easily exceed several million dollars. Financial support for such projects could
not be obtained easily through traditional funding agencies as the risk/reward ratio was considered too high. Furthermore, substantial infrastructure for sample acquisition, microdissection of tumor from normal tissue, nucleic acid preparation, high throughput automated sequencing, and computational analysis of the resulting data were essential. Few institutions were equipped to address these challenges. In response, the National Cancer Institute in the United States (in partnership with the National Human Genome Research Institute) and an international group known as the International Cancer Genome Consortium (ICGC) launched large-scale efforts to sequence the complete genomes of thousands of cancers. By 2010 the U.S. effort (called The Cancer Genome Atlas [TCGA]) had completed an analysis of glioblastoma32 and ovarian cancer and had launched efforts in lung, kidney, endometrial, and other cancers. The international consortium had committed to sequencing 25,000 tumors representing 50 different cancer subtypes.33 Both groups stipulated immediate release of all sequence information to the research community free of charge so that the entire scientific community could learn from the data. These grand-scale projects were possible due to a dramatic decline in sequencing costs. Discussions just a few years earlier about which genes (or regions of genes) to include in such projects were no longer necessary. The cost of complete exome (all exons from all genes in the human genome—not just kinases) and complete genome (all DNA) resequencing had fallen to about $5,000 per sample.
not be obtained easily through traditional funding agencies as the risk/reward ratio was considered too high. Furthermore, substantial infrastructure for sample acquisition, microdissection of tumor from normal tissue, nucleic acid preparation, high throughput automated sequencing, and computational analysis of the resulting data were essential. Few institutions were equipped to address these challenges. In response, the National Cancer Institute in the United States (in partnership with the National Human Genome Research Institute) and an international group known as the International Cancer Genome Consortium (ICGC) launched large-scale efforts to sequence the complete genomes of thousands of cancers. By 2010 the U.S. effort (called The Cancer Genome Atlas [TCGA]) had completed an analysis of glioblastoma32 and ovarian cancer and had launched efforts in lung, kidney, endometrial, and other cancers. The international consortium had committed to sequencing 25,000 tumors representing 50 different cancer subtypes.33 Both groups stipulated immediate release of all sequence information to the research community free of charge so that the entire scientific community could learn from the data. These grand-scale projects were possible due to a dramatic decline in sequencing costs. Discussions just a few years earlier about which genes (or regions of genes) to include in such projects were no longer necessary. The cost of complete exome (all exons from all genes in the human genome—not just kinases) and complete genome (all DNA) resequencing had fallen to about $5,000 per sample.
Rounding Out the Treatment of Myeloproliferative Disorders: JAK2 and Myelofibrosis
Taken together with the BCR-ABL translocation in chronic myeloid leukemia and FIP1L1-PDGFRα in HES, the discovery of JAK2
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