Many of the first generation TKIs are not very selective, often having many kinase targets in addition to the target tyrosine kinase [31
]. In large part this stems from the fact that appropriate counterscreens were not available for the majority of protein kinases at the time when these inhibitors were being developed. In addition, the technology for developing selective kinase inhibitors has improved significantly, particularly with the use of structure- and fragment-based design methods. Nevertheless, off-target specificities did not preclude the development of these first generation TKIs as drugs, and indeed a combination of on-target and off-target effects may partly underlie the effectiveness of these TKIs in some circumstances, particularly when there is redundancy in the tyrosine kinases that are activated in a tumor. The multikinase specificity of imatinib has been put to great use in the treatment of tumors that depend on mutationally activated c-KIT or PDGFRα RTKs, such as gastrointestinal stromal tumors (GIST). Moreover, a virtue is now being made of broad inhibitor specificity through the development of multikinase inhibitors.
The effectiveness of TKIs in cancer therapy provides strong support for the idea that individual tumors can be dependent on a single activated tyrosine kinase. However, there is also emerging evidence for redundancy among driver RTKs in cancer, such that inhibition of one tyrosine kinase may result in upregulation or activation of a second RTK that can serve the same function [34
]. This suggests that the use of combinations of TKIs or multikinase inhibitors may be more effective. The “addiction” of cancers to specific signaling pathways is now being explored by screening the sensitivity of large numbers of tumor cell lines to approved protein kinase inhibitors [36
]. In general, the most sensitive lines prove to contain activating mutations in the target kinase. For instance, the NSCLC cell lines most sensitive to erlotinib have activating EGFR mutations, and the melanoma, thyroid, and colorectal carcinoma lines sensitive to a RAF inhibitor have mutant B-RAF [36
]. Kinome-wide resequencing studies on large numbers of tumor cell lines and primary tumors have become another avenue for pinpointing mutant kinases driving the tumor phenotype that have not previously been implicated in cancer [37
]. The use of phosphoproteomic analysis of P.Tyr-containing peptides also has the potential to uncover activated tyrosine kinases in cancer [40
]. Finally, kinome-wide siRNA screens of tumor cell lines harboring defined oncogenic mutations for protein kinase dependence are yielding interesting and distinct patterns of kinase dependency that highlight essential signaling pathways and provide kinase targets for inhibition that may not necessarily be mutant in the cancer [41
Despite the success of the first generation of TKIs , resistance to the drugs is a frequent event, and in the great majority of cases this is found to be due to point mutations within the catalytic domain that render it resistant to inhibition. This has been particularly well worked out in the case of imatinib-resistant CML, where a large number of point mutations that render BCR-ABL resistant to imatinib has been identified through sequencing [46
]. The most common is T315I, a mutation in the so-called gatekeeper residue, which blocks imatinib binding to the catalytic cleft because the bulkiness of the Ile side chain results in a steric clash. Mutation of the equivalent residue in EGFR, T790, to Met also commonly underlies the resistance of NSCLC with mutant EGFR [47
] to treatment with the EGFR inhibitor erlotinib [49
]. The propensity of tumors to generate resistant variants due to their genetic instability has demanded the generation of TKIs that can inhibit the mutant forms (e.g. sunitinib for imatinib-resistant GIST, and dasatinib and nilotinib for imatinib-resistant CML). In addition, prospective studies are now routinely carried out to determine what sort of mutations will render a target tyrosine kinase resistant to an inhibitor that is in clinical trials, so that a head start on developing second generation inhibitors can be made. In practice, it may prove beneficial to start therapy with a combination of inhibitors targeting the same tyrosine kinase, analogous to triple therapy for HIV, to reduce the chances of the tumor developing a resistant form of the kinase.
Looking forward, we can expect continuing development of second generation TKIs to circumvent the problem of resistance to first generation compounds. Allosteric inhibitors, which interact with sites outside the kinase domain catalytic cleft, are likely to become commonplace. In this regard, small molecule inhibitors that interact with the extracellular domains of RTKs and prevent dimerization are likely to be developed. Molecular diagnosis of individual tumors for activated tyrosine kinases will be used to define which TKIs to use for treatment. For therapeutic purposes, we need to learn how to use TKIs in the right combination with other signal transduction inhibitors, and with chemotherapeutic drugs, and to optimize dosing and scheduling to treat cancers more effectively. It will also be critical to elucidate the rules underlying the crosstalk and feedback mechanisms that lead to activation of redundant tyrosine kinase pathways upon inhibition of a single tyrosine kinase in cancer. Undoubtedly, TKIs will also be approved for use in the treatment of other types of disease, e.g. inflammatory and immune-related conditions, although this raises issues about the safety profiles appropriate for long term use of TKIs.