Charles L. Sawyers
INTRODUCTION
In 2001, the first tyrosine-kinase inhibitor imatinib was approved for clinical use in chronic myeloid leukemia. 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 progress in this field over ~15 years, with the focus on the concepts underlying this paradigm shift as well as the considerable challenges that remain (Table 22.1). 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) receptor tyrosine kinases covered here have also been successfully targeted by monoclonal antibodies that engage these proteins at the cell surface. These drugs, 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 to 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 Abelson murine leukemia (ABL) kinase inhibitor imatinib was the recognition that tumor cells acquire exquisite dependence on the breakpoint cluster region protein 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, the survival of which is regulated by a complex array of growth factors and interactions with the bone marrow microenvironment. Although BCR-ABL clearly gives this cell a growth advantage that, over years, results in the clinical phenotype of chronic myeloid leukemia, 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, just like their normal, nontransformed neighbors. Thus, it seemed more likely that, by shutting down the driver oncogene, BCR-ABL inhibitors might halt the progression of chronic myeloid leukemia but not eliminate the preexisting tumor cells. In fact, chronic myeloid leukemia (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 still remains to be defined, the notion of finding an Achilles’ heel for each cancer continues to captivate the cancer research community and has spawned 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 (BCR-ABL, KIT, HER2)
From the beginning, clinical trials of imatinib were restricted to patients with Philadelphia chromosome–positive chronic myeloid leukemia. 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 patients in the early phase studies.3 It was obvious that 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. As we will see, it took nearly 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 us in the face. Foremost among them is the notion that tumors with a 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. This has also led to a new paradigm at the regulatory level of drug approval requiring codevelopment of a companion diagnostic (a molecularly based diagnostic test that reliably identifies patients with the mutation) with the new drug.
After chronic myeloid leukemia, 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 (PDGF) receptor.4,5 Serendipitously, imatinib inhibits both KIT and the PDGF receptor; 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 histologic 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, which 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 and 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 for the 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.
The Serendipity of Unexpected Clinical Responses: EGFR in Lung Cancer
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 those who never smoked, 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.10–12 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.
But that was 2004, when the prevailing approach to cancer drug development was an empiric one originally developed (with great success) for cytotoxic agents. Typically, small numbers of patients with different cancers were treated in all comer phase I 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 disease-specific phase II trial would justify a randomized phase III registration trial, where the typical endpoint for drug approval is time to progression or survival. Cytotoxics were 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. (Note: The use of the past tense here is intentional. As we will see later in this chapter, nearly all cancer drug development today is based on selecting patients with a certain molecular profile.)
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 chemotherapy-refractory, 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 III studies with survival endpoints.13 The sponsors of both drugs, therefore, conducted phase III registration studies in patients with chemotherapy-refractory, 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 but study amendments could have been considered.) 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 because the two drugs have highly similar chemical structures and biologic properties. Perhaps the most important difference 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 because 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 were clearly documented at lower doses.
In parallel with the single-agent phase III trials in chemotherapy-refractory patients, both gefitinib and erlotinib were studied as an 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.16–18 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 fact that EGFR mutations are present in only 10% to 15% of patients (i.e., those likely to benefit) provided a logical explanation. The clinical signal from those whose tumors had EGFR mutations was likely diluted out 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 also for missteps in deciding that the EGFR genotype should drive treatment selection. Perhaps the most egregious error came from a retrospective analysis of tumors from patients treated on the BR.21 trial, which concluded that EGFR mutations did not predict for a survival advantage.19 (EGFR gene amplification was associated with survival, but only in a 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. Fortunately, recent advances in DNA mutation detection, using massively parallel next-generation sequencing technology, have largely eliminated this concern. These new platforms are now being used in the clinical setting.
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.20Conversely, 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.
A Mix of Science and Serendipity: PDGF Receptor–Driven 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 CML, 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 PDGF receptor). ABL and KIT were quickly excluded, but the PDGF receptor α (PDGFR-α) gene was targeted by an interstitial deletion that fused the upstream FIP1L1 gene to PDGFR-α23 FIP1L1-PDGFR-α is a constitutively active tyrosine kinase, analogous to BCR-ABL, and is 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-PDGFR-α 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-PDGFR-β 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 PDGFB ligand (not the receptor). COL1A-PDGFB is oncogenic through autocrine stimulation of the normal PDGF receptor in these tumor cells. Patients with dermatofibrosarcoma protuberans respond to imatinib therapy because it targets the PDGF receptor, 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 a 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 cancers.27 Another was the discovery of mutations in the JAK2 kinase in nearly all patients with polycythemia vera, as well as a significant fraction of patients with myelofibrosis and essential thrombocytosis.28–30 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 phosphatase and tensin homolog (PTEN) 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 in the follow paragraphs.
Each of these important discoveries—BRAF, JAK2, and PIK3CA—came from relatively small efforts (less than 100 tumors) and 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 the high cost of DNA sequencing using the Sanger method. 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 because the risk/reward was considered too high. Furthermore, substantial infrastructure for sample acquisition, microdissection of the tumors from normal tissue, nucleic acid preparation, high throughput automated sequencing, and computational analysis of the resulting data was 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. In parallel, next-generation sequencing technologies resulted in massive reductions in cost, allowing a more comprehensive analysis of much larger numbers of tumors. At the time of this writing, the US effort (called The Cancer Genome Atlas [TCGA]) had reported data on 29 different tumor types (https://tcga-data.nci.nih.gov/tcga/). The international consortium has committed to sequencing 25,000 tumors representing 50 different cancer subtypes.32 Both groups have enforced immediate release of all sequence information to the research community free of charge so that the entire scientific community can learn from the data. This policy enabled pan cancer mutational analyses that give an overall view of the genomic landscape of cancer, serving as a blueprint for the community of cancer researchers and drug developers.33,34
Rounding Out the Treatment of Myeloproliferative Disorders: JAK2 and Myelofibrosis
Taken together with the BCR-ABL translocation in CML and FIP1L1-PDGFR-α in HES, the discovery of JAK2 mutations in polycythemia, essential thrombocytosis, and myelofibrosis provided a unifying understanding of myeloproliferative disorders as diseases of abnormal kinase activation. The JAK family kinases are the primary effectors of signaling through inflammatory cytokine receptors and, therefore, had been considered compelling targets for anti-inflammatory drugs. But the JAK2 mutation discovery immediately shifted these efforts toward developing JAK2 inhibitors for myeloproliferative disorders. Because most patients have a common JAK2 V617F mutation, these efforts could rapidly focus on screening for activity against a single genotype. Progress has been rapid. Myelofibrosis was selected as the initial indication (instead of essential thrombocytosis or polycythemia vera) because the time to registration is expected to be the shortest. Currently, ruxolitinib is approved for myelofibrosis based on shrinkage in spleen size as the primary endpoint. Clinical trials in essential thrombocytosis and polycythemia vera (versus hydroxyurea) are ongoing. Other JAK2 inhibitors are also in clinical development.
BRAF Mutant Melanoma: Several Missteps Before Finding the Right Inhibitor
As with JAK2 mutations in myeloproliferative disorders, the discovery of BRAF mutations in patients with melanoma launched widespread efforts to find potent BRAF inhibitors. One early candidate was the drug sorafenib, which had been optimized during drug discovery to inhibit RAF kinases. (Sorafenib also inhibits vascular endothelial growth factor (VEGF) receptors, which led to its approval in kidney cancer, as discussed later in this chapter.) Despite the compelling molecular rationale for targeting BRAF, clinical results of sorafenib in melanoma were extremely disappointing and reduced enthusiasm for pursuing BRAF as a drug target.35 In hindsight, this concern was completely misguided. Sorafenib dosing is limited by toxicities that preclude achieving serum levels in patients that potently inhibit RAF, but are sufficient to inhibit VEGF receptors. In addition, patients were enrolled without screening for BRAF mutations in their tumors. Although the frequency of BRAF mutations in melanoma is high, the inclusion of patients without the BRAF mutation diluted the chance of seeing any clinical signal. In short, the clinical evaluation of sorafenib in melanoma was poorly designed to test the hypothesis that BRAF is a therapeutic target. The danger is that negative data from such clinical experiments can slow subsequent progress. It is critical to know the pharmacodynamic properties of the drug and the molecular phenotype of the patients being studied when interpreting the results of a negative study.
The fact that RAF kinases are intermediate components of the well-characterized RAS/ mitogen-activated protein (MAP) kinase pathway (transducing signals from RAS to RAF to MEK to ERK) raised the possibility that tumors with BRAF mutations might respond to inhibitors of one of these downstream kinases (Fig. 22.1). Preclinical studies revealed that tumor cell lines with BRAF mutation were exquisitely sensitive to inhibitors of the downstream kinase MEK.36 (Sorafenib, in contrast, does not show this profile of activity.37 Thus, proper preclinical screening would have revealed the shortcomings of sorafenib as a BRAF inhibitor.) Curiously, cell lines with a mutation or amplification of EGFR or HER2, which function upstream in the pathway, were insensitive to MEK inhibition. Even tumor lines with RAS mutations were variably sensitive. In short, the preclinical data made a strong case that MEK inhibitors should be effective in BRAF mutant melanoma, but not in other subtypes. The reason that HER2, EGFR, and RAS mutant tumors were not sensitive to MEK inhibitors is explained, at least in part, by the existence of negative feedback loops that modulate the flux of signal transduction through MEK.38
In parallel with the generation of these preclinical findings, clinical trials of several MEK inhibitors were initiated. Patients with various cancers were enrolled in the early studies, but there was a strong bias to include melanoma patients. Significant efforts were made to demonstrate MEK inhibition in tumor cells by measuring the phosphorylation status of the direct downstream substrate ERK using an immunohistochemical analysis of biopsies from patients with metastatic disease. Phase I studies of the two earliest compounds in clinical development (PD325901 and AZD6244) documented reduced phospho-ERK staining at multiple dose levels in several patients for whom baseline and treatment biopsies were obtained.39,40 (In the following, we will learn that these pharmacodynamic studies, while well intentioned, were not quantitative enough to document the magnitude of MEK inhibition in these patients.) Furthermore, clinical responses were observed in a few patients with BRAF mutant melanoma. Armed with this confidence, a randomized phase II clinical trial of AZD6244 was conducted in advanced melanoma, with the chemotherapeutic agent temozolomide (which is approved for glioblastoma) as the comparator arm. (The clinical development of PD325901 was discontinued because of safety concerns about ocular and neurologic toxicity.) Disappointingly, patients receiving AZD6244 had no benefit in progression-free survival when compared to temozolomide-treated patients, raising further concerns about the viability of BRAF as a drug target.41 A closer examination of the data revealed that clinical responses were, indeed, seen in patients receiving AZD6244. The fact that BRAF mutation status was not required for study entry likely diminished the clinical signal in the AZD6244 arm, a lesson learned from the EGFR inhibitor trials in lung cancer. Indeed, a different MEK inhibitor, trametinib, received FDA approval in 2013 based on activity in melanoma patients with the BRAF mutation.42
All doubts about BRAF as a target vanished in 2009 to 2010 when dramatic clinical responses were observed with a novel BRAF inhibitor vemurafenib (PLX4032). Like sorafenib, this compound was optimized to inhibit RAF, but with an additional focus on mutant BRAF. Vemurafenib differs dramatically from sorafenib because it potently inhibits BRAF without the additional broad range of activities that sorafenib has against other kinases like the VEGF receptor.43 The greater selectivity of vemurafenib relative to sorafenib resulted in a much greater tolerability, such that it could be given at high doses while avoiding significant toxicity. The early days of vemurafenib clinical development were plagued by challenges in maximizing the oral bioavailability of the drug.44 Consequently, the initial phase I clinical trial was temporarily halted to develop a novel formulation (i.e., the coingredients in the drug capsule or tablet that improve solubility and absorption through the gastrointestinal tract). Much higher serum levels were obtained in patients who received the new vemurafenib formation and, shortly thereafter, complete and partial responses were observed in about 80% of the melanoma patients with B-RAF mutant tumors. Strikingly, no activity was observed in patients whose tumors were wild type for BRAF.45,46 The data were so compelling that vemurafenib was immediately advanced to a phase III registration trial. Similarly impressive responses in BRAF mutant melanoma patients were observed with a second potent RAF inhibitor dabrafenib,47 providing further proof that BRAF is a important cancer target.
The vemurafenib and dabrafenib data also provide insight into why sorafenib and the early MEK inhibitor trials failed to demonstrate activity. One lesson is the critical importance of achieving adequate target inhibition. Clinical responses with vemurafenib were observed only after the drug was reformulated to achieve substantially higher serum levels. Reductions in phospho-ERK staining (as documented by immunohistochemistry) were documented in the earlier trials but, in retrospect, the assays were not sensitive enough to distinguish between modest (~50%) kinase inhibition versus more complete BRAF or MEK inhibition. Efficacy in preclinical models is significantly improved using doses that give >80% inhibition, and the human trial data suggest that this degree of pathway blockade is also required for a high clinical response rate.46 Collectively, these experiences illustrate the critical need for quantitative pharmacodynamic assays to measure target inhibition early in clinical development. A second lesson is the importance of genotyping all patients for mutation or amplification of the relevant drug target. Not only does this ensure that a sufficient number of patients with the biomarker of interest are included in the study, but also that the results provide compelling evidence early in clinical development in support (or not) of the preclinical hypothesis.
Getting It Right: ALK and Lung Cancer
The development of the ALK inhibitor crizotinib (PF-02341066) illustrates how an unexpected signal obtained in a small number of patients can quickly shift a program in an entirely new direction with a high probability of success. The key ingredient is this story is a familiar one—a strong molecular hypothesis backed up by clinical response data in a small number of carefully selected patients. Crizotinib emerged from a drug discovery program at Pfizer that was focused on finding inhibitors of the MET receptor tyrosine kinase and entered the clinic with this target as its lead indication.48 As we previously learned with imatinib, essentially all kinase inhibitors have activity against other targets (so called off-target activities), which can sometimes prove to be advantageous. Off-target activities are typically discovered by screening compounds against a large panel of kinases to establish profiles of relative selectivity against the intended target. Off-target activity, potency, and pharmaceutical properties (bioavailability, half-life) are all factors that influence the decision of which compound to advance to clinical development. The primary off-target activity of crizotinib is against the ALK tyrosine kinase.
ALK was first identified as a candidate driver oncogene in 1994 through the cloning of the t(2,5) chromosomal translocation associated with anaplastic large cell lymphoma, which creates the nucleophosmin/anaplastic lymphoma kinase (NPM-ALK) fusion gene.49 This discovery, together with the demonstration that NPM-ALK causes lymphoma in mice, made a compelling case for ALK as a drug target in this disease. But there was limited interest in developing ALK inhibitors because this particular lymphoma subtype is rare and most commonly found in children. (Companies are generally reluctant to develop drugs solely for pediatric indications because of complexities related to dose selection and additional regulatory guidelines. Efforts to streamline this development process are underway, such as the Creating Hope Act, which provides new incentives for companies to pursue pediatric indications.) In 2007, a different ALK fusion gene called EML4-ALK was discovered in a small fraction of patients with lung adenocarcinoma, with an estimated frequency of 1% to 5%.50 This discovery did not immediately capture the attention of drug developers, but several academic groups who had already begun testing lung cancer patients seen at their institutions for EGFR mutations simply added an EML4-ALK fusion test to the screening panel. Astute clinical investigators participating in the phase I trial of crizotinib, which was designed to include patients with a broad array of advanced cancers, were aware of the off-target ALK activity and enrolled several lung cancer patients with EML4-ALK fusions in the study. These patients had remarkably dramatic responses.51 This serendipitous finding in a few ALK-positive patients was confirmed in a larger cohort, resulting in a strongly positive pivotal phase III study in ALK-positive lung cancer, just 2 years after the discovery of the EML4-ALK fusion.52 Crizotinib is also being evaluated in other diseases associated with genomic alterations in ALK, including large-cell anaplastic lymphoma, neuroblastoma,53 and inflammatory myofibroblastic sarcoma.54
Extending the Model to RET Mutations in Thyroid Cancer: Clinical Responses, But Why?
Subsets of patients with papillary or medullary thyroid cancer have activating mutations or translocations targeting the RET tyrosine-kinase receptor, raising the question of whether RET inhibitors might have a role in this disease.55Although no drugs specifically designed to inhibit RET have entered the clinic, four compounds with off-target activity against RET (vandetanib, sorafenib, motesanib, and cabozantinib) have all shown single-agent activity in thyroid cancer studies.56–60 Vandetanib and cabozantinib are currently approved in medullary thyroid cancer based on improved progression-free survival in phase III registration trials.61,62 Because all four compounds also inhibit VEGF receptor, it is unclear whether the clinical benefit observed in these studies is explained by inhibition of RET, VEGF receptor, or both. Unlike the crizotinib trials in ALK-positive lung cancer, enrollment in these registration studies was not restricted to patients with RET mutations. In addition to the fact that thyroid cancer patients are not routinely screened for these mutations, the primary reason for including all comers in these studies is that clinical responses are observed in a larger fraction of patients than can be accounted for based on the suspected frequency of an RET mutation. Responses in patients without RET mutation (if they occur) might be explained by mutations in other genes in the RAS-MAP kinase pathway such as BRAF or HRAS, which are found in a substantial fraction of patients and typically do not overlap with RET alterations.55 Clearly, detailed genotype/response correlations, as demonstrated in lung cancer and melanoma, will clarify the role of these mutations in predicting the response to these drugs. Thyroid cancer is also a compelling indication for the BRAF and MEK inhibitors discussed previously in melanoma.
FLT3 Inhibitors in Acute Myeloid Leukemia: Did the Genomics Mislead Us?
Shortly after the success of imatinib, the receptor tyrosine–kinase FLT3 emerged as a compelling drug candidate based on the presence of activating mutations in about one-third of patients with acute myeloid leukemia.63 Laboratory studies documented that FLT3 alleles bearing these mutations, which occur as internal tandem duplications (ITD) of the juxtamembrane domain or a point mutation in the kinase domain, function as driver oncogenes in mouse models, giving phenotypes analogous to BCR-ABL.64 As with RET in thyroid cancer, no compounds had been specifically optimized to target FLT3, but several drugs with off-target FLT3 activity were redirected to acute myeloid leukemia (AML). Disappointingly, the first three of the compounds tested (midostaurin, lestaurtinib, and sunitinib) showed only marginal single-agent activity in relapsed AML patients, even in those with FLT3 mutations.65–67Despite the strong molecular rationale for FLT3 as a driver lesion, questions were raised about the viability of FLT3 as a drug target. Pharmacodynamic studies showed evidence of FLT3 kinase inhibition in tumor cells, but the magnitude and duration of these effects were difficult to quantify, raising the possibility of inadequate target inhibition.65 Indeed, the dose of all three compounds was limited by toxicities believed to be independent of FLT3. A more pessimistic interpretation was that FLT3, although presumably important for the initiation of AML, was no longer required for tumor maintenance due to the accumulation of additional driver genomic alterations. If true, even a complete FLT3 blockade with a highly selective inhibitor would be expected to fail. But this view was not supported by the fact that clinical responses were observed in the somewhat analogous situation of single-agent ABL kinase inhibitor treatment of CML in blast crisis, where BCR-ABL is just one of many additional genomic alterations that contribute to disease progression, yet complete remissions are observed in many patients.
Despite this pessimism about FLT3 as a viable drug target, several drugs are now advancing toward drug registration trials. Midostaurin, one of the early compounds that showed disappointing single-agent activity in relapsed AML, is being evaluated in a randomized phase III trial in newly diagnosed AML combined with standard induction chemotherapy. A single-arm phase II study showed higher and more durable remission rates in FLT3 mutant patients when compared to historical controls.68 The second compound, quizartinib (AC220), is a next-generation FLT3 inhibitor with greater potency and specificity and with single-agent activity in FLT3 mutant relapsed AML—precisely the population where midostaurin and others failed.69,70 The fact that some responder patients have relapsed with drug-resistant gatekeeper mutations in the FLT3 kinase domain provides formal proof that FLT3 is the relevant target.71 Assuming these compounds prove successful in AML, it will be important to examine their activity in the rare cases of pediatric acute lymphoid leukemia associated with FLT3 mutation. Although the jury is still out on FLT3 inhibitors, the failure of early compounds in AML is reminiscent of the failures of early RAF and MEK inhibitors in melanoma. Collectively, these examples emphasize the importance of using optimized compounds to test a molecularly based hypothesis in patients and to focus enrollment on those patients with the relevant molecular lesion.
Kidney Cancer: Targeting the Tumor and the Host With Mammalian Target of Rapamycin and VEGF Receptor Inhibitors
A recurring theme in this chapter is the critical role of driver kinase mutations in guiding the development of kinase inhibitors. Ironically, several kinase inhibitors have been approved for kidney cancer over the past 5 years in a tumor type with no known kinase mutations. The most common molecular alteration in kidney cancer is a loss of function in the Von Hippel-Lindau (VHL) tumor suppressor gene, resulting in the activation of the hypoxia inducible factor68 pathway.72 As a consequence of VHL loss, which normally targets hypoxia-inducible factor (HIF) proteins for proteasomal degradation, HIF-1α and HIF-2α are constitutively active transcription factors that function as oncogenes through activation of an array of downstream target genes. Among these is the angiogenesis factor VEGF, which is secreted by HIF-expressing cells and promotes the development and maintenance of tumor neovasculature. HIF-mediated secretion of VEGF by tumor cells likely explains the highly vascular histopathology of clear cell renal carcinoma. All three currently approved angiogenesis inhibitors (the monoclonal antibody bevacizumab targeting VEGF and the kinase inhibitors sorafenib and sunitinib targeting or its receptor VEGF receptor) have single-agent clinical activity in clear cell carcinoma.73–75 The high specificity of bevacizumab for VEGF leaves little doubt that the activity of this drug is explained by antiangiogenic effects. In contrast, the off-target activities of sorafenib and sunitinib include several kinases expressed in kidney tumor cells, stroma, and inflammatory cells (PDGFR, RAF, RET, FLT3, and others). Interestingly, the primary effect of bevacizumab in kidney cancer is disease stabilization, whereas sorafenib and sunitinib have substantial partial response rates. This raises the question of whether the superior antitumor activity of the VEGF receptor kinase inhibitors is due to the concurrent inhibition of other kinases. However, partial responses rates with next-generation VEGF receptor inhibitors (axitinib, pazopanib, and tivozanib), all of which have greater potency and selectivity for the VEGF receptor, are similarly high, and reinforce the importance of the VEGF receptor as the critical target in kidney cancer.76–78 Pazopanib is approved for advanced kidney cancer, whereas axitinib is approved as second-line therapy.
Two inhibitors of the mammalian target of rapamycin (mTOR) kinase (temsirolimus and everolimus) are also approved for advanced renal cell carcinoma.79,80 Both temsirolimus and everolimus are known as rapalogs because both are chemical derivatives of the natural product sirolimus (rapamycin). Sirolimus was approved more than 10 years ago to prevent graft rejection in transplant recipients based on its immunosuppressive properties against T cells. Sirolimus also has potent antiproliferative effects against vascular endothelial cells and, on that basis, is incorporated into drug-eluting cardiac stents to prevent coronary artery restenosis following angioplasty.81 Rapalogs differ from all the other kinase inhibitors discussed in this chapter in that they inhibit the kinase through an allosteric mechanism rather than by targeting the mTOR kinase domain. Because rapalogs also inhibit the growth of cancer cell lines from different tissues of origin, clinical trials were initiated to study their potential role as anticancer agents in a broad range of tumor types. Based on responses in a few phase I patients with different tumor types (including kidney cancer), exploratory phase II studies were conducted in several diseases. Single-agent activity of temsirolimus was observed in a phase II kidney cancer study,82 then confirmed in a phase III registration trial.79 The phase III everolimus trial, which was initiated after temsirolimus, was noteworthy because clinical benefit was demonstrated in patients who had progressed on the VEGF receptor inhibitors sorafenib or sunitinib.80
In parallel with the empirical clinical development of rapalogs, various laboratories explored the molecular basis for mTOR dependence in cancer cells. mTOR functions at the center of a complex network that integrates signals from growth factor receptors and nutrient sensors to regulate cell growth and size (Fig. 22.2). It does so, in part, by controlling the translation of various mRNAs with complex 5′ untranslated regions into protein. mTOR exists in two distinct complexes known as TOR complex 1 (TORC1) and TORC2. Rapalogs only inhibit the TORC1 complex, which is largely responsible for downstream phosphorylation of targets such as S6K1/2 and 4EBP1/2 that regulate protein translation.83 The TORC2 complex contributes to the activation of AKT by phosphorylating the important regulatory serine residue S473 and is unaffected by rapalogs.
Two hypotheses have emerged to explain the clinical activity of rapalogs in kidney cancer. The antiproliferative activity of these compounds against endothelial cells suggests an antiangiogenic mechanism, which is consistent with the clinical activity of the VEGF receptor inhibitors. But rapalogs also inhibit the growth of kidney cancer cell lines in laboratory models where the effects on tumor angiogenesis have been eliminated. Interestingly, mRNAs for HIF1/2 are among those whose translation is impaired by rapalogs, and this effect has been implicated as the primary mechanism of rapalog activity in kidney cancer xenograft models.84 As with the VEGF receptor inhibitors, a detailed molecular annotation of tumors from responders and nonresponders will shed light on these issues.
Other Indications for mTOR Inhibitors: Breast Cancer and Tuberous Sclerosis Complex Mutant Cancers
Two other indications for mTOR have emerged, both based on fundamental insights from laboratory studies but from quite different angles. Preclinical studies of estrogen receptor (ER) therapy in breast cancer suggested that phosphatidylinositol 3-kinase (PI3K) pathway activation may be a mechanism of resistance and that this resistance could be prevented or overcome by combined treatment with ER-based drugs and rapalogs such as everolimus. Based on evidence that some women with progressive disease while receiving the aromatase inhibitor letrozole have clinical benefit from the addition of everolimus, randomized trials were initiated comparing everolimus + exemestane to exemestane alone (called BOLERO-2), or everolimus + tamoxifen to tamoxifen alone (called TAMRAD). Both studies demonstrated substantial improvements in time to progression in women with metastatic breast cancer who had already failed one aromatase inhibitor,85,86 resulting in FDA approval of the everolimus/exemestane combination. Evidence of cross-talk between the PI3K pathway and hormone receptor signaling (ER in breast cancer, androgen receptor in prostate cancer) provides a molecular rationale for the clinical benefit of combination therapy and is currently under investigation in metastatic prostate cancer.87
Yet another indication for rapalog therapy emerged from the genetics of children with tuberous sclerosis caused by a loss of function mutations in tuberous sclerosis complex 1 (TSC1) or TSC2, which encode the proteins hamartin and tuberin that function in the PI3K signaling pathway just upstream of mTOR. Based on laboratory studies showing that TSC1- or TSC2-deficient cells are exquisitely sensitive to rapalogs, a clinical trial was conducted in tuberous sclerosis patients with benign subependymal giant-cell astrocytomas (SEGA) that showed tumor shrinkage in 21 of 28 patients.88 This genetic dependence on mTOR in tumors with tuberous sclerosis complex (TSC) loss has also been observed in bladder cancer. In a remarkable example of the power of comprehensive DNA sequencing to provide insight into rare clinical phenotypes, investigators examined the tumor genome of the single complete responder patient on a phase II trial of everolimus in bladder cancer and discovered somatic mutations in TSC2 as well as a second gene, NF2, that also controls mTOR activity.89 This plus other examples of how a retrospective genomic analysis of extraordinary responders has led to a national effort to capture these cases, as well as prospective clinical trials of patients with the relevant tumor genotype regardless of histology (called basket trials).
It is unclear why rapalogs have failed in other tumor types. One explanation is the concurrence of PI3K pathway mutations with alterations in other pathways that mitigate sensitivity to rapalogs. Another possibility is the disruption of negative feedback loops regulated by mTOR that inhibit signaling from upstream receptor tyrosine kinases. Rapalogs paradoxically increase signaling through PI3K due to loss of this negative feedback. A primary consequence is increased AKT activation, which signals to an array of downstream substrates that can enhance cell proliferation and survival (other than TORC1, which remains inhibited by rapalog) (see Fig. 22.2). This problem might be overcome by combining rapalogs with an inhibitor of an upstream kinase in the feedback loop, such as HER kinases or the insulinlike growth factor receptor (IGFR), to block this undesired effect of rapalogs on PI3K activation.90
DIRECTLY TARGETING THE PI3K PATHWAY
Mutations or copy number alterations (e.g., amplification or deletion of oncogenes or tumor suppressor genes) in PI3K pathway genes (PIK3CA, PIK3R1, PTEN, AKT1, and others) are among the most common abnormalities in cancer. Consequently, intensive efforts at many pharmaceutical companies have been devoted to the discovery of small-molecule inhibitors targeting kinases in the PI3K pathway. Inhibitors of PI3K, AKT, and ATP-competitive (rather than allosteric) inhibitors of mTOR that target both the TORC1 and TORC2 complex are all in clinical development. Phase I clinical trials have, in general, established that the pathway can be efficiently targeted without serious toxicity other than easily manageable effects on glucose metabolism (which is anticipated based on the importance of PI3K signaling in insulin signaling). Unfortunately, there has been no evidence to date of dramatic single-agent clinical activity with any of these agents, although early results with PI3K alpha selective inhibitor BYL719 in PIK3CA mutant breast cancer appear promising.91
However, the first approval of a direct PI3K inhibitor in cancer is likely to come in chronic lymphocytic leukemia and in lymphoma, but not on the basis of tumor genomics. Normal and malignant B cells are dependent on PI3K delta as well as Bruton tyrosine kinase (BTK) for proliferation and survival, raising the possibility that inhibitors of these kinases might be broadly active in B-cell malignancies. Concerns about toxicity on normal B cells were alleviated, in part, by the earlier clinical success of the CD20 antibody rituximab in lymphoma, which also eliminates normal circulating B cells, but without significant clinical sequelae. The first such PI3K delta inhibitor, idelalisib, has shown impressive activity in indolent non-Hodgkin lymphoma as a single agent and in relapsed chronic lymphocytic leukemia when given in combination with rituximab. The BTK inhibitor ibrutinib, following a similar clinical development path, was recently approved as second-line therapy for chronic lymphocytic leukemia and for mantle cell lymphoma.92,93
COMBINATIONS OF KINASE INHIBITORS TO INDUCT RESPONSE AND PREVENT RESISTANCE
Preclinical studies indicate that combinations of kinase inhibitors are required to realize their full potential as anticancer agents. The most common rationale is to address the problem of concurrent mutations in different pathways that alleviate dependence on a single-driver oncogene. The best examples are cancers with mutations in both the RAS/MAP kinase pathway (RAS or BRAF) and the PI3K pathway (PIK3CA or PTEN). In mouse models, such doubly mutant tumors fail to respond to single-agent treatment with either an AKT inhibitor or a MEK inhibitor. However, combination treatment can give dramatic regressions.94 Similarly, genetically engineered mice that develop KRAS-driven lung cancer respond only to combination therapy with a PI3K inhibitor and a MEK inhibitor.95 To date, clinical trials combining different PI3K pathway and RAS/MAP kinase pathway inhibitors have been challenging due to toxicities associated with continuous, concurrent PI3K and RAS/MAP kinase pathway inhibition.
Many of the tumor types discussed in this chapter do respond to treatment with a single-agent kinase, but relapse despite continued inhibitor therapy. Research into the causes of “acquired” kinase inhibitor resistance has revealed two primary mechanisms: (1) novel mutations in the kinase domain of the drug target that preclude inhibition, or (2) bypass of the driver kinase signal by activation of a parallel kinase pathway. In both cases, the solution is combination therapy to prevent the emergence of resistance. An elegant demonstration of this approach comes from CML where resistance to imatinib is primarily caused by mutations in the BCR-ABL kinase domain.96,97 The second-generation ABL inhibitors dasatinib and nilotinib are effective against most imatinib-resistant BCR-ABL mutants and were initially approved as single-agent therapy for imatinib-resistant CML.98,99 Very recently, both drugs have proven superior to imatinib in the upfront treatment of CML due to increased potency and fewer mechanisms of acquired resistance.100–102 However, one BCR-ABL mutation called T315I is resistant to all three drugs. The third-generation ABL kinase inhibitor ponatinib blocks T315I and showed activity in a phase II clinical trial that included CML patients with the T315I mutation,103 resulting in FDA approval. However, subsequent reports of severe vascular occlusive events, such as stroke and heart failure, led to withdrawal from the market, followed by approval for restricted use in T315I-mutant patients. Analogous approaches are ongoing in other diseases such as EGFR-mutant lung cancer, where acquired resistance to the frontline kinase inhibitor is also associated with mutations in the target kinase.104,105 Promising clinical results have been reported with irreversible EGFR inhibitors such as CO-1686 and AZD9291.
The clinical development of kinase inhibitor combinations to prevent acquired resistance is relatively straightforward. Because the frontline drug is already approved, success would be determined by an improvement in response duration using the combination. The situation is more complex when two experimental compounds (e.g., a PI3K pathway inhibitor and a MEK inhibitor) are combined, neither of which shows significant single-agent activity. Older regulatory guidelines required a four-arm study that compared each single agent to the combination and to a control group in order to obtain approval of the combination. Recognizing that this design could discourage drug developers as well as patients from moving forward because it requires a large sample size, the FDA has issued new guidelines for the development of novel combinations that require a two-arm registration study comparing the combination to standard of care http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM236669.pdf. A more challenging issue may be dose optimization and dose schedule that is needed to safely combine two investigational drugs. Much like the development of combination chemotherapy several decades ago, it may be important to select compounds with nonoverlapping toxicities to allow for sufficient doses of each drug to be achieved.
SPECULATIONS ON THE FUTURE ROLE OF KINASE INHIBITORS IN CANCER MEDICINE
The role of genomics in predicting a response to kinase inhibitor therapy is now irrefutable. As the number of kinase driver mutations continues to grow, the field is likely to move away from the current strategy of a companion diagnostic for each drug. Rather, comprehensive mutational profiling platforms that query each tumor for hundreds of potential cancer mutations are more likely to emerge as the diagnostic platform. The number of directly actionable mutations (meaning the presence of a mutation defines a treatment decision supported by clinical trial data) remains low, but this number will undoubtedly grow. In addition, it is becoming apparent that many patients have rare mutations (defined as rare in that histologic tumor type) but are, in theory, actionable. Because these examples are unlikely to be formally evaluated in clinical trials, many centers have opened basket studies (with eligibility based solely on mutation profile) to capture these cases with some reports of remarkable success.
More effort must be devoted to manipulating the dose and schedule of kinase inhibitor therapy to maximize efficacy and minimize toxicity. To date, all kinase inhibitors have been developed based on the assumption that a 24/7 coverage of the target is required for efficacy. Consequently, most compounds are optimized to have a long serum half-life (12 to 24 hours). Phase II doses are then selected based on the maximum tolerated dose determined with daily administration. But a recent clinical of the ABL inhibitor dasatinib in CML indicates that equivalent antitumor activity can be achieved with intermittent therapy.106 By giving larger doses intermittently, higher peak drug concentrations were achieved that resulted in equivalent and possibly superior efficacy.107 Similar results were observed in laboratory studies of EGFR inhibitors in EGFR-mutant lung cancer. Clinically robust, quantitative assays of target inhibition are needed to hasten progress in this area.
Although the focus of this chapter is kinase inhibitors, the themes developed here should apply broadly to inhibitors of other cancer targets. Inhibitors of the G-protein coupled receptor smoothened (SMO) in patients with metastatic basal cell carcinoma or medulloblastoma establish that the driver mutation hypothesis extends beyond kinase inhibitors. SMO is a component in the Hedgehog pathway, which is constitutively activated in subsets of patients with basal cell carcinoma and medulloblastoma due to mutations in the Hedgehog ligand-binding receptor Patched-1. Treatment with the SMO inhibitor vismodegib led to impressive responses in basal cell carcinoma and medulloblastoma patients whose tumors had Patched-1 mutations,108,109 resulting in FDA approval. Other novel cancer targets are emerging from cancer genome sequencing projects. Somatic mutations in the Krebs cycle enzyme isocitrate dehydrogenase (IDH1/2) were found in subsets of patients with glioblastoma, AML, chondrosarcoma, and cholangiocarcinoma,110–112 and the first IDH2 inhibitor has entered clinical trials in leukemia. Mutations in enzymes involved in chromatin remodeling, such as the histone methyltransferase EZH2, have been reported in lymphoma and have spurred the ongoing development of EZH2 inhibitors.113,114 Inhibitors of another histone methyltransferase DOT1L, which is required for the maintenance of mixed lineage leukemia (MLL) fusion leukemias, are also in clinical development.115,116 Kinase inhibitors are just the first wave of molecularly targeted drugs ushered in by our understanding of the molecular underpinnings of cancer cells. There is much more to follow.
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