Article Figures & Data
Figures
- Figure 1.
Ponatinib potently inhibits a broad spectrum of KIT primary activating and secondary resistance mutants in vitro and in vivo. IC50 values (nmol/L) of imatinib, sunitinib, regorafenib, and ponatinib in Ba/F3 cells harboring (A) native or primary mutant KIT alone (green bars), (B) exon 11 (Δ557–558) + ATP pocket secondary mutants (blue bars), and (C) exon 11 (Δ557–558) + A-loop secondary mutants (red bars). The cell lines were treated with increasing concentrations of the drug for 3 days followed by cell viability assessment using the MTT assay. Data are shown as mean ± SD from 3 separate experiments. D, in vivo efficacy of ponatinib, imatinib, and sunitinib in subcutaneous tumor models using Ba/F3 KIT-mutant cells. Imatinib (300 mg/kg) was used as a comparator to ponatinib (30 mg/kg) in all 4 models. On the basis of in vitro potencies, sunitinib was included as a second comparator in 2 models (Δ557–558/V654A and Δ557–558/T670I) in which imatinib was expected to be nonefficacious, and a lower dose of ponatinib (10 mg/kg) was also tested in the Δ557–558 and Δ557–558/D816H models. Tumor-bearing animals were treated once daily by oral gavage with vehicle or the indicated doses of drug for 12 days. Mean tumor volume and SEM are plotted. Each treatment group was compared with the relevant vehicle group using 1-way ANOVA, with statistical significance (P < 0.05) indicated by an asterisk.
- Figure 2.
Secondary resistance mutants identified in the presence of KIT inhibitors. Resistant cells were recovered from N-ethyl-N-nitrosourea–treated Ba/F3 KIT Ex11 (Δ557–558) cells and cultured with imatinib, sunitinib, regorafenib, or ponatinib at the indicated concentrations. Each bar represents the relative frequency of the indicated KIT kinase domain secondary mutation, based on next-generation sequencing data. (Reported mutation frequencies are a composite of both mutation incidence and cell number.) ATP pocket residues are underlined in red and A-loop residues in blue. Mutagenesis data are shown from a representative experiment; similar results were obtained in 3 separate studies.
- Figure 3.
Co-crystal structure of KIT bound with ponatinib. A, crystal structure of ponatinib in complex with the native KIT kinase domain. Ponatinib is shown in gold, side chains of C673 (hinge region) and other key amino acids referred to in the text in green, the A-loop in cyan, and the juxtamembrane (JM) domain in magenta. B, left, W557 of the KIT JM domain (green) occupies the DFG pocket in the apo form. Right, ponatinib (gold) displaces W557 and the JM domain upon binding. C, the impact of V654A mutation on ponatinib binding. Left, the green dashed lines indicate van der Waals contacts between V654 (green) and ponatinib (gold). Right, the mutation of valine to alanine (magenta) results in a loss of all van der Waals contacts with ponatinib. D, illustration of the ability of ponatinib to accommodate the space requirements of T670I gatekeeper mutant. The increase in steric bulk upon mutation from T670 (green, left) to I670 (magenta, right) is accommodated by the triple bond of ponatinib (gold).
- Figure 4.
Ponatinib activity in patient-derived KIT-driven tumor models. A, KIT phosphorylation and downstream signaling were evaluated by immunoblot in GIST430, GIST430/654, and GIST-1 PDX-implanted animals treated with a single oral dose of vehicle (V), 30 mg/kg ponatinib (PO), or 300 mg/kg imatinib (IM), n = 3 per group. B, in vivo efficacy of ponatinib, imatinib, sunitinib, and regorafenib in GIST-1 PDX model. Tumor-bearing animals were treated once daily by oral gavage with vehicle or the indicated dose of drug for the indicated dosing period. Mean tumor volume and SEM are plotted. The vehicle used for ponatinib and sunitinib is shown (citrate buffer); nearly identical tumor growth was observed for the vehicles used for imatinib (water) and regorafenib (NMP/PEG; data not shown). Statistical significance, calculated using one-way ANOVA (P < 0.05) in which each treatment group (day 28) was compared to its vehicle control, is indicated by an asterisk. Data are shown for all groups until fewer than 8 of the original 10 mice in each group remained. In the vehicle and imatinib groups, mice were sacrificed when tumors became too large. In the sunitinib treatment group, multiple mice were sacrificed because of >20% body weight loss.
- Figure 5.
Single-agent ponatinib is active in heavily pretreated patients with TKI-resistant GIST. Representative CT scans of 3 patients with KIT exon 11-mutant GIST before and after treatment with 30-mg ponatinib for 4 weeks. Each patient was heavily pretreated with imatinib, sunitinib, and regorafenib. A, patient 1: ponatinib induced regression and cyst-like transformation of multiple metastatic lesions. B, patient 2: ponatinib induced moderate responses in multiple lesions. C, patient 3: no response to ponatinib treatment. Red arrows highlight areas of tumor growth, whereas yellow arrows indicate tumor response.
Tables
- Table 1.
Ponatinib IC50 values (nmol/L) in cell lines established from tumor biopsies in patients with GIST
Cell line KIT status Ponatinib Imatinib Sunitinib Regorafenib GIST882 K642E 31 173 54 503 GIST430 Δ560–576 12 61 68 191 GIST430/654 Δ560–576/V654A 159 1,204 90 1,001 GIST430/654/680 Δ560–576/V654A/N680K 330 >5,000 1,314 4,969 GIST-T1 Δ560–578 5 30 15 110 GIST-T1/670 Δ560–578/T670I 8 >5,000 48 249 GIST-T1/816 Δ560–578/D816E 23 604 3,111 395 GIST-T1/829 Δ560–578/A829P 16 1,201 1,168 934 GIST48/820 V560D/D820A 34 413 587 164 GIST48B KIT-independent 806 >5,000 >5,000 >5,000 GIST226 KIT-independent 2,807 >5,000 3,856 >5,000
Supplementary Data
Files in this Data Supplement:
- Supplementary Figures - Supplementary Figures. Figure S1. Chemical structures of imatinib, sunitinib, regorafenib and ponatinib. Figure S2. Expression and activation of KIT in engineered Ba/F3 cells. Figure S3. Ponatinib inhibits the phosphorylation of exon 11primary activating, and secondary resistant mutant forms of KIT. Figure S4. Secondary mutants reduce ponatinib potency in Ex9 ins and V560D cell lines. Figure S5. Illustration of the optimal fit of ponatinib to KIT. Figure S6. Impact of compound treatment on KIT signaling in GIST-derived cell lines.
- Supplementary Tables - Supplementary Tables. Table S1. In Vitro Kinase IC50 Values (nM) for Native and Mutant Recombinant KIT. Table S2. Summary of Ba/F3 KIT Cell Lines Generated in this Study. Table S3. Summary of IC50 Viability Values in Ba/F3 KIT Cells. Table S4. Summary of KIT Driven Ba/F3 In Vivo Studies.
- Supplementary methods, patient information and figure legends - Supplementary methods, patient information and figure legends
Volume 20, Issue 22
