Sequential Kinase Inhibitor Therapy in CML Patients Can Select for Cells Harboring Compound BCR-ABL Kinase Domain Mutations with Increased Oncogenic Potency: Rationale for Early Combination Therapy of ABL Kinase Inhibitors.
Authors: Shah Neil P. , Skaggs Brian , Branford Susan , Hughes Timothy P. , Nicoll John M. , Paquette Ronald L. , Sawyers Charles L. 
Published: April 2020

Abstract
A critical question in the targeted therapy era relates to whether treatment outcomes will be optimized by sequential or combinatorial use of targeted agents. Selection for CML cells with BCR-ABL kinase domain mutations is the main mechanism responsible for loss of response to imatinib. Dasatinib is an ABL tyrosine kinase inhibitor that has activity against nearly all imatinib-resistant mutations and is approved for the treatment of imatinib-resistant and -intolerant BCR-ABL-associated leukemias.
Acquired clinical resistance to sequential use of dasatinib following imatinib failure has been observed. We analyzed the BCR-ABL kinase domain at the time of relapse in 15 patients who lost an initial response to dasatinib, and found evolution of a total of three new mutations at the time of relapse in all cases. The highly resistant BCR-ABL/T315I mutation was detected in 11 cases. The four remaining cases were associated with the evolution of novel mutations (V299L, 3 cases; T315A, 1 case). V299L was also detected in a fourth case that had also evolved T315I. These three dasatinib-resistant mutations were part of a small number of amino acid substitutions previously isolated in a preclinical mutagenesis screen for dasatinib resistance-conferring BCR-ABL mutations. While the T315I mutation is highly resistant to imatinib, V299L and T315A retain sensitivity to imatinib in vitro and have not been previously described in imatinib-resistant cases, raising the potential utility of imatinib rechallenge in select dasaitinib-resistant cases.
A significant finding of our studies is the evolution of five unique “compound” mutations (i.e. greater than one mutation on a DNA strand) in the BCR-ABL kinase domain of patients treated sequentially with imatinib and dasatinib. It is noteworthy that although the imatinib-sensitive V299L and T315A mutations evolved in five cases, they were detected in the context of a pre-existing imatinib-resistant mutation in three of these cases, and these cases are therefore unlikely to respond to rechallenge with IM. The T315A mutation was detected in the context of 2 pre-existing IM-resistant mutations (M244V/L364I). Interestingly, in bone marrow transformation assays, the clinically-identified dasatinib-resistant M244V/L364I/T315A mutation was more potently oncogenic than non-mutated BCR-ABL, in contrast to the baseline imatinib resistant M244V/L364I, which like T315A in isolation, was less potent than native BCR-ABL
Our studies of CML cases resistant to sequential kinase inhibitor therapy reinforce BCR-ABL kinase domain mutation as the predominant mechanism of resistance to kinase inhibitor therapy, and provide evidence that compound mutations acquired as a result of sequential therapy can not only limit further therapeutic options, but also create more biologically aggressive isoforms of BCR-ABL. Together, these findings provide a strong rationale for early treatment of CML with combinations of kinase inhibitors that have the capacity to collectively prevent selection of resistant kinase domain mutations.