Recommendations are based on activity comparisons factors, such as protein binding, they are indeed clinically useful. quality of life. Open in a separate window Figure 1 Tyrosine kinase inhibitors (TKIs) approved for the treatment of chronic myeloid leukemia. Uramustine (a) The crystal structure of the ABL1 kinase domain is shown in complex with the indicated TKI. Highlighted residues indicate mutations that confer resistance to the indicated TKI genotype, providing a prime example of personalized therapy in Uramustine oncology. Here, we discuss TKI therapy for CML to illustrate the challenges of molecularly targeted cancer therapy, focusing on therapy individualization, the role of clonal evolution and complexity in therapy response and resistance, and how the lessons learned from CML may be applied to TKI therapy in other types of cancer. Development of BCR-ABL1 TKIs for CML Most patients are diagnosed in CML-CP, during which the myeloid cell compartment is expanded but cellular differentiation is maintained . Without effective therapy, CML-CP inexorably progresses to blast phase CML (CML-BP), a disease that resembles an acute leukemia, with complete block of terminal differentiation and a poor prognosis. Murine models indicate that BCR-ABL1 is required and sufficient to induce CML-CP, whereas diverse Uramustine additional mutations have been implicated in progression to CML-BP (Table?1) [3,5C16]. Table 1 Mutations associated with CML-BP assays based on culturing cells that express randomly mutagenized BCR-ABL1 in the presence of TKIs are remarkably accurate in predicting clinically relevant BCR-ABL1 resistance mutations and contact points between TKIs and the kinase domains. Mutagenesis is achieved either by initial expression of a BCR-ABL1 plasmid in a mutagenic bacterial strain or by exposing the BCR-ABL1-expressing cells to N-nitroso-N-methylurea (ENU). Despite the fact that activity is dependent on multiple additional factors, including bioavailability, achievable plasma concentrations, transmembrane transport and protein binding, the drug sensitivity of cell lines (typically the pro-B cell line BaF/3, engineered to express BCR-ABL1 mutants in comparison to the native BCR-ABL1 kinase) is generally correlated with clinical activity (Figure?3). This allows rational TKI selection on the basis of the patients genotype, and provides an example of how molecular knowledge can aid the personalization of cancer therapy. Open in a separate window Figure 3 Activities of Uramustine imatinib, bosutinib, dasatinib, nilotinib, and ponatinib against mutated forms of BCR-ABL1. Half maximal inhibitory concentration (IC50) values for cell proliferation of the indicated TKIs are shown against BCR-ABL1 single mutants. The color gradient demonstrates the IC50 sensitivity for each TKI relative to its activity against cells expressing native BCR-ABL1. Note that clinical activity is also dependent on additional factors, such as the drug concentrations achieved in the plasma of patients. Adapted with permission from Redaelli molecule) is inferred if the percentages of mutant alleles combined, based on their peak height relative to that of the native sequence, exceed 100%. If the combined mutant alleles are less than 100%, Sanger sequencing cannot distinguish between compound mutations and polyclonal mutations (that is, multiple BCR-ABL1 mutant clones). A widely used method to ascertain that two mutations localize to the same allele is shotgun cloning of PCR products followed by sequencing of individual colonies; however, long-range NGS may provide a less tedious approach in the future . Colony sequencing has been used to demonstrate linear clonal evolution in several patients who developed multidrug-resistant compound mutant clones . Interestingly, the likelihood that an additional mutation is silent rather than missense increases with the total number of mutations in the BCR-ABL1 molecule (Figure?4). This suggests that the fitness of the BCR-ABL1 kinase must ultimately be compromised by the acquisition of successive missense mutations, leading to evolutionary CYFIP1 dead ends. From a therapeutic standpoint, this is good news as it suggests that mutational escape of the primary target kinase is not unlimited. As the impact on kinase fitness of two mutations in the.