Molecular dynamics reveal BCR-ABL1 polymutants as a unique mechanism of resistance to PAN-BCR-ABL1 kinase inhibitor therapy

Abstract

The acquisition of mutations within the BCR-ABL1 kinase domain is frequently associated with tyrosine kinase inhibitor (TKI) failure in chronic myeloid leukemia. Sensitive sequencing techniques have revealed a high prevalence of compound BCR-ABL1 mutations (polymutants) in patients failing TKI therapy. To investigate the molecular consequences of such complex mutant proteins with regards to TKI resistance, we determined by cloning techniques the presence of polymutants in a cohort of chronic-phase patients receiving imatinib followed by dasatinib therapy. The analysis revealed a high frequency of polymutant BCR-ABL1 alleles even after failure of frontline imatinib, and also the progressive exhaustion of the pool of unmutated BCR-ABL1 alleles over the course of sequential TKI therapy. Molecular dynamics analyses of the most frequent polymutants in complex with TKIs revealed the basis of TKI resistance. Modeling of BCR-ABL1 in complex with the potent pan-BCR-ABL1 TKI ponatinib highlighted potentially effective therapeutic strategies for patients carrying these recalcitrant and complex BCR-ABL1 mutant proteins while unveiling unique mechanisms of escape to ponatinib therapy.

Keywords: compound mutation; ponatinib resistance.

Fig. 1.

Mutated and unmutated BCR-ABL1 in CML clones during sequential tyrosine kinase inhibitor therapy. (A) Dynamics of unmutated BCR-ABL1–bearing clones. Patients without cytogenetic response during dasatinib therapy after imatinib failure bore a lower proportion of unmutated clones compared with those who achieved at least a partial cytogenetic response (P = 0.01), suggesting exhaustion of unmutated BCR-ABL1–bearing clones in patients not responding to sequential TKI therapy. (B) Mapping of the clinically relevant mutations found onto the BCR-ABL1 protein in complex with dasatinib. The different colors reflect the density of mutations found in each region of the kinase: the darker the color the higher the density of mutations in that specific region. Color code: light blue (9%): A-loop (residues 379–398); sky blue (13%): T315-F317 region; blue (16%): P-loop (residues 242–261); navy blue (17%): catalytic cleft (residues 351–359); dark blue (18%): hotspot region (residues 295–312). (C) Survival of patients carrying mutations in the hotspot 295–312 region. Patients carrying mutations at T315I or at the 295–312 region exhibit a shorter overall survival compared with those carrying other BCR-ABL1 mutations (P = 0.03).

Fig. 2.

In silico modeling of ponatinib binding to BCR-ABL1 polymutants. (A) (Top) Details of the binding site of unmutated BCR-ABL1 in complex with ponatinib as obtained from equilibrated MD simulation snapshots. The protein backbone is portrayed as a transparent gray ribbon; the main residues involved in drug interactions are shown as labeled colored sticks. Hydrogen bonds are shown as green lines. (Middle) Superposition of the binding site of unmutated BCR-ABL1 (red) and the BCR-ABL1^T315I (purple) in complex with ponatinib. In the two complexes, the drug is depicted in red (unmutated) and purple (T315I) sticks and balls, respectively. (Bottom) Superposition of the binding site of unmutated BCR-ABL1 (red) and the BCR-ABL1^V299L/F317L double mutant (black) in complex with ponatinib. In the two complexes, the drug is depicted in red (unmutated) and black (F317L/V299L) sticks and balls, respectively. (B) MD snapshots of dasatinib (Upper) and ponatinib (Lower) bound BCR-ABL1^T315I/F317L. The mutant I315 and L317 are depicted as orange and cyan sticks, respectively. Note the marked difference in steric clash (golden areas in both panels), accounting for the higher affinity of ponatinib for the double-mutant protein. Hydrogen atoms, water molecules, and ions are omitted for clarity. (C) Sensitivity of a series of BCR-ABL1 proteins with single point mutations (black bars) or dual mutants (red bars) against ponatinib, based on calculated IC₅₀. Dotted blue line represents the half-maximal inhibitory concentration (IC₅₀) of ponatinib against BCR-ABL1^T315.

Fig. 3.

Effect of single and dual BCR-ABL1 kinase mutations on Ba/F3 cellular sensitivity to tyrosine kinase inhibitors. (A) The dual mutant T315I/F359V induces a rearrangement of the BCR-ABL1 binding pocket, with the addition of the F359V mutation resulting in loss of drug–protein surface complementarity and formation of a cavity (Lower, in blue), that promotes a weaker protein/inhibitor interaction with respect to the unmutated BCR-ABL1 protein (Upper, in red). (B) Activity of imatinib, dasatinib, and ponatinib against unmutated BCR-ABL1 and BCR-ABL1 carrying T315I, T315I/V299L, T315I/F317L, and T315I/F359V mutations in Ba/F3-based cellular assays. (C) Stat5 and CrkL phosphorylation was examined in lysates obtained from Ba/F3 cells carrying unmutated or mutated BCR-ABL1 proteins upon exposure to increasing concentrations of ponatinib for 4 h. Samples were analyzed by immunoblot analysis with antibodies against phospho-Stat5, phospho-CrkL, and actin (loading control).