Abstract B277: Polymutant BCR-ABL1 proteins during chronic myeloid leukemia therapy: Novel mechanisms of resitance from clinical, in vitro, and in silico evidence.
Authors: Pricl Sabrina , Gibbons Don L. , Posocco Paola , Laurini Erik , Giabbai Barbara , Storici Paola , Fermeglia Maurizio , Sun Hunshi , Cortes Jorge , Talpaz Moshe , Donato Nicholas , Quintas-Cardama Alfonso 
Published: August 2014

Abstract
The acquisition of mutations within the BCR-ABL1 kinase domain is frequently associated to imatinib failure among patients with chronic myeloid leukemia (CML). Typically, patients failing imatinib undergo sequential therapy with second line tyrosine kinase inhibitors (TKIs) such as nilotinib or dasatinib. To investigate the prevalence of BCR-ABL1 mutations and the mechanisms of acquired TKI resistance during sequential therapy, we used a sensitive cloning technique in a cohort of chronic-phase patients failing imatinib therapy that subsequently received dasatinib. The analysis revealed that most patients failing imatinib carry BCR-ABL1 mutations, many with polymutant BCR-ABL1 alleles, and that there is progressive exhaustion of the pool of unmutated BCR-ABL1 alleles over the course of treatment. Detailed modeling of the structural effects on the ABL1 kinase domain of the various mutations and of clinically available/experimental TKIs against some of the most frequent polymutants found in our patient cohort indicates that the accumulation of more than one BCR-ABL1 mutation within the same allele promotes a higher than expected level of TKI resistance compared to that of each single mutation alone. Some polymutants exhibited levels of resistance to imatinib/dasatinib remarkably higher than those of the highly resistant T315I mutation, suggesting that the generation of polymutant BCR-ABL1 alleles represents a powerful mechanism of escape for CML cells exposed to TKI-induced selection pressure. Notably, the complexity of the polymutants is limited, suggesting that, in addition to increasing TKI resistance, the successive accumulation of single mutations within the same clone may compromise its fitness, the latter being a function of the number and type of single mutants involved in any given polymutant. Strategies to limit the generation of complex mutants and clinical resistance may require the use of frontline therapy with TKI combinations or with novel TKIs that cover a broad range of mutations and therefore may prevent the emergence of resistance. In addition to providing an understanding for the molecular basis of clinical TKI failure in our patient cohort, we modeled the activity of the novel TKI ponatinib against the most frequently encountered polymutants. Although all the polymutants were predicted to be highly resistant to imatinib and dasatinib, ponatinib was predicted to maintain a notable affinity for most clinically relevant BCR-ABL1 single mutants and some of the polymutants detected in our patient cohort. Ponatinib was able to accommodate the structural changes induced by these complex mutants, which suggest the possibility of polymutant kinase inhibition at doses frequently within a clinically achievable range. These findings were validated in cell-based biochemical assays, thus supporting the clinical applicability of in silico modeling as a means to predict the TKI approach with the highest probability of clinical success. It is worth noting, however, that some polymutants examined required ponatinib concentrations unlikely to be reached in humans without excessive toxicity, thus anticipating potential mechanisms of escape to ponatinib therapy through selection and expansion of clones carrying highly TKI resistant complex BCR-ABL1 mutant proteins.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B277.
Citation Format: Sabrina Pricl, Don L. Gibbons, Paola Posocco, Erik Laurini, Barbara Giabbai, Paola Storici, Maurizio Fermeglia, Hunshi Sun, Jorge Cortes, Moshe Talpaz, Nicholas Donato, Alfonso Quintas-Cardama. Polymutant BCR-ABL1 proteins during chronic myeloid leukemia therapy: Novel mechanisms of resitance from clinical, in vitro, and in silico evidence. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B277.