BCR-ABL1 compound mutations combining key kinase domain positions confer clinical resistance to ponatinib in Ph chromosome-positive leukemia

Abstract

Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-ABL1 single mutants in Philadelphia chromosome-positive (Ph(+)) leukemia, including the recalcitrant BCR-ABL1(T315I) mutant. However, emergence of compound mutations in a BCR-ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1 compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib, dasatinib, ponatinib, rebastinib, and bosutinib. T315I-inclusive compound mutants confer high-level resistance to TKIs, including ponatinib. In vitro resistance profiling was predictive of treatment outcomes in Ph(+) leukemia patients. Structural explanations for compound mutation-based resistance were obtained through molecular dynamics simulations. Our findings demonstrate that BCR-ABL1 compound mutants confer different levels of TKI resistance, necessitating rational treatment selection to optimize clinical outcome.

Figure 1. Clinically Reported BCR-ABL1 Compound Mutations are Centered on 12 Key Positions

(A) Crystal structure of the ABL1 kinase domain in complex with imatinib (PDB entry 2HYY). Twelve key positions accounting for most clinical BCR-ABL1 TKI resistance, including compound mutation-based resistance, are highlighted (orange; T315 is in red). The phosphate-binding (yellow) and activation loops (green) are indicated. (B, C) Key resistance positions (orange) in clinically reported T315I-inclusive (B) and non-T315I BCR-ABL1 compound mutants (C). Substitutions at non-key positions are in gray. 1, Shah et al., 2007; 2, Khorashad et al., 2008; 3, Stagno, et al., 2008; 4, Kim et al., 2010; 5, Kim, D.W. et al., Blood 2010 abstract 3443; 6, Khorashad et al., 2013; 7, Smith, C.C., et al., Blood 2011, abstract 3752; 8, Soverini, et al. 2013; 9, Cortes, et al. 2013; 10, Hochhaus et al., 2013; *, the current study. See also Figure S1 and Table S1.

Figure 2. Single and Compound BCR-ABL1 Mutants Exhibit Differential TKI Sensitivity in Ba/F3 Cells

(A, B) Cell proliferation IC₅₀ values of TKIs against BCR-ABL1 single mutants (A), T315I-inclusive (B, left) and non-T315I (B, right) compound mutants. Mean IC₅₀ values are plotted. Blue lines denote average steady-state plasma concentration of each TKI in patients taking the standard daily dose: imatinib (400 mg; 3377 nM) (Peng et al., 2004; Gozgit et al., Blood 2013, abstract 3992), nilotinib (400 mg twice daily; 2754 nM) (Kantarjian et al., 2006; Gozgit et al., Blood 2013, abstract 3992), dasatinib (100 mg; 27 nM) (Gozgit et al., Blood 2013, abstract 3992; Talpaz et al., 2006), ponatinib (15, 30, and 45 mg/day doses are represented; 35, 84, and 101 nM respectively) (Cortes et al., 2012; Gozgit et al., Blood 2013, abstract 3992), rebastinib (150 mg twice daily; 350 nM) (Chan et al., 2011; Eide et al., 2011) and bosutinib (500 mg; 287 nM) Gozgit et al., Blood 2013, abstract 3992). (C) Heat map of TKI IC₅₀s for single and compound mutants. I315M (see Figure 6) and T315I/E453K (see Figure 5) are included for comparison. A color gradient from green (sensitive) to yellow (moderately resistant) to red (highly resistant) denotes the IC₅₀ sensitivity to each TKI: imatinib (green: <1000 nM; yellow: 1000-4000 nM; red: >4000 nM); nilotinib (green: <200 nM; yellow: 200-1000 nM; red: >1000 nM); dasatinib (green: <25 nM; yellow: 25-150 nM; red: >150 nM); ponatinib (green: <25 nM; yellow: 25-150 nM; red: >150 nM); rebastinib (green: <100 nM; yellow: 100-1000 nM; red: >1000 nM); bosutinib (green: <150 nM; yellow: 150-1000 nM; red: >1000 nM). (D, E) BCR-ABL1 tyrosine phosphorylation (Y393) immunoblot analysis of T315I-inclusive (D) and non-T315I (E) compound mutants. See also Table S2.

Figure 3. Molecular Dynamics Simulations of ABL1^Y253H/E255V Provide a Structural Explanation for the Ineffectiveness of Ponatinib Compared to Dasatinib

(A) Structural alignment of native ABL1 kinase (PDB entry 3IK3 bound to ponatinib; orange) with the ABL1^Y253H/E255V mutant kinase (blue). Ponatinib is shown in wireframe. Positions within 4 Å of ponatinib were designated for structural alignment using only the C^α atoms. Native positions Y253 and E255 (green) and mutated positions H253 and V255 (red) are shown as spheres. The region of the backbone (P-loop, C-helix and DFG loop) that showed major conformational change is highlighted in either orange (native) or blue (Y253H/E255V). (B) Positions of selected residues in the ponatinib site of native ABL1 (orange) and ABL1^Y253H/E255V (cyan). (C) Dasatinib was docked to a representative model of the ABL1^Y253H/E255V mutant structure using MD simulation. Key residues involved in binding of dasatinib are shown (cyan). The Y253H substitution establishes a new hydrogen bond with dasatinib, in addition to the existing T315-dasatinib hydrogen bond (red dashed lines). The chemical structure of dasatinib is shown using a space-filling wireframe representation. (D) Computational docking of dasatinib (black) and ponatinib (gray) into the mutant ABL1^Y253H/E255V kinase domain (Glide XP; Schrödinger, 2012). (E) Fold-change in cellular IC₅₀ for BCR-ABL1^Y253H/E255V relative to native BCR-ABL1 for dasatinib (black) and ponatinib (gray). Error bars represent +/- SEM. See also Figure S2.

Figure 4. BCR-ABL1^E255V/T315I Exhibits Severely Compromised TKI Binding and is Detected at Clinical Relapse on Ponatinib

(A) Summary of BCR-ABL1 cloning and sequencing for Patient #38 (Ph⁺ ALL) at baseline (upper) and EOT (lower) by cloning and sequencing individual amplicons. Conventional sequencing results and prior TKIs are shown. Light red bars represent mutations detected at key positions; dark red fractions of bars indicate additional mutations at other positions. For bars designated “None”, light and dark red fractions represent native BCR-ABL1 and all mutations not occurring at key positions, respectively. In this patient, a dominant, pan-TKI resistant E255V/T315I mutant was detected at the time of ponatinib failure. (B) Molecular dynamics comparison of E255V/T315I (blue; V255 in red) and T315I mutants (orange; E255 in green). (C) Structural realignments in E255V/T315I (cyan) compared to the T315I mutant (orange). See also Figure S3 and Table S6.

Figure 5. Clinical Emergence of T315I-Inclusive Compound Mutants Can Lead to Relapse

(A) Summary of BCR-ABL1 cloning and sequencing of individual clones for Patient #36 (CML-BP) at baseline (upper) and EOT (lower). Conventional sequencing results and prior TKIs are shown. Bar graphs are as in Figure 4. (B) Fold-change in ponatinib Ba/F3 cellular IC₅₀ values for BCR-ABL1^F359V and BCR-ABL1^T315I/F359V compared to native BCR-ABL1. (C) Summary of BCR-ABL1 cloning and sequencing for Patient #12 (CML-AP). Samples were analyzed as in (A) at baseline (upper), while on ponatinib (middle), and at EOT (lower). (D) Fold-change in ponatinib Ba/F3 cellular IC₅₀ values for BCR-ABL1^T315I and BCR-ABL1^T315I/E453K compared to native BCR-ABL1. All error bars represent +/- SEM. See also Table S7.

Figure 6. Acquired I315M Point Mutation-Based Ponatinib Failure in a Patient Harboring T315I at Onset of Therapy

(A) Summary of BCR-ABL1 cloning and sequencing for Patient #22 (Ph⁺ ALL) at baseline (upper), longitudinally on ponatinib therapy (middle), and at EOT (lower) by cloning and sequencing individual clones. Conventional sequencing results and prior TKIs are shown. Bar graphs are as in Figure 4. (B) Fold-change in ponatinib Ba/F3 cellular IC₅₀ values for BCR-ABL1^I315M and BCR-ABL1^T315I compared to native BCR-ABL1. Error bars represent +/- SEM. (C) Ponatinib (in wireframe) in complex with ABL1^T315I (orange); ABL1^I315M (blue) is superimposed. Structural alignment of the T315I and I315M mutants was performed as in Figure 3. (D) M315 penetrates deeply into the ponatinib site, and structural adjustments at positions 269, 290, 293, 317, 359 and 381 also disfavor ponatinib binding. See also Figure S4 and Table S8.

Figure 7. Certain Non-T315I Compound Mutants Lead to Clinical Relapse, While Others Are Susceptible to Ponatinib

(A) Summary of BCR-ABL1 cloning and sequencing for Patient ^#37 (Ph⁺ ALL) at baseline (upper) and EOT (lower) by cloning and sequencing individual amplicons. Conventional sequencing results and prior TKIs are shown. Bar graphs are as in Figure 4. (B) Summary of BCR-ABL1 cloning and sequencing for Patient #34 (CML-AP) at baseline (upper) and while responding to ponatinib (lower). (C) Summary of BCR-ABL1 cloning and sequencing for Patient #23 (CML-CP) at baseline (upper) and EOT (lower). (D) Summary of BCR-ABL1 cloning and sequencing for Patient #27 (CML-CP) at baseline (upper) and while responding to ponatinib (lower). See also Table S9.