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. 2014 Nov 15;20(22):5745-5755.
doi: 10.1158/1078-0432.CCR-14-1397. Epub 2014 Sep 19.

Ponatinib Inhibits Polyclonal Drug-Resistant KIT Oncoproteins and Shows Therapeutic Potential in Heavily Pretreated Gastrointestinal Stromal Tumor (GIST) Patients

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Free PMC article

Ponatinib Inhibits Polyclonal Drug-Resistant KIT Oncoproteins and Shows Therapeutic Potential in Heavily Pretreated Gastrointestinal Stromal Tumor (GIST) Patients

Andrew P Garner et al. Clin Cancer Res. .
Free PMC article

Abstract

Purpose: KIT is the major oncogenic driver of gastrointestinal stromal tumors (GIST). Imatinib, sunitinib, and regorafenib are approved therapies; however, efficacy is often limited by the acquisition of polyclonal secondary resistance mutations in KIT, with those located in the activation (A) loop (exons 17/18) being particularly problematic. Here, we explore the KIT-inhibitory activity of ponatinib in preclinical models and describe initial characterization of its activity in patients with GIST.

Experimental design: The cellular and in vivo activities of ponatinib, imatinib, sunitinib, and regorafenib against mutant KIT were evaluated using an accelerated mutagenesis assay and a panel of engineered and GIST-derived cell lines. The ponatinib-KIT costructure was also determined. The clinical activity of ponatinib was examined in three patients with GIST previously treated with all three FDA-approved agents.

Results: In engineered and GIST-derived cell lines, ponatinib potently inhibited KIT exon 11 primary mutants and a range of secondary mutants, including those within the A-loop. Ponatinib also induced regression in engineered and GIST-derived tumor models containing these secondary mutations. In a mutagenesis screen, 40 nmol/L ponatinib was sufficient to suppress outgrowth of all secondary mutants except V654A, which was suppressed at 80 nmol/L. This inhibitory profile could be rationalized on the basis of structural analyses. Ponatinib (30 mg daily) displayed encouraging clinical activity in two of three patients with GIST.

Conclusion: Ponatinib possesses potent activity against most major clinically relevant KIT mutants and has demonstrated preliminary evidence of activity in patients with refractory GIST. These data strongly support further evaluation of ponatinib in patients with GIST.

Figures

Figure 1
Figure 1. Ponatinib Potently Inhibits a Broad Spectrum of KIT Primary Activating and Secondary Resistance Mutants In Vitro and In Vivo
IC50 values (nM) of imatinib, sunitinib, regorafenib and ponatinib in Ba/F3 cells harboring (A) native or primary mutant KIT alone (green bars), (B) Ex11 (Δ557-558) + ATP pocket secondary mutants (blue bars) and (C) Ex11 (Δ557-558) + A-loop secondary mutants (red bars). The cell lines were treated with increasing concentrations of drug for three days followed by cell viability assessment using the MTT assay. Data are shown as mean ± SD from three 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 four models. Based on in vitro potencies, sunitinib was included as a second comparator in two models (Δ557-558/V654A and Δ557-558/T670I) in which imatinib was expected to be non-efficacious, 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 to the relevant vehicle group using one way ANOVA, with statistical significance (p< 0.05) indicated by an asterisk.
Figure 1
Figure 1. Ponatinib Potently Inhibits a Broad Spectrum of KIT Primary Activating and Secondary Resistance Mutants In Vitro and In Vivo
IC50 values (nM) of imatinib, sunitinib, regorafenib and ponatinib in Ba/F3 cells harboring (A) native or primary mutant KIT alone (green bars), (B) Ex11 (Δ557-558) + ATP pocket secondary mutants (blue bars) and (C) Ex11 (Δ557-558) + A-loop secondary mutants (red bars). The cell lines were treated with increasing concentrations of drug for three days followed by cell viability assessment using the MTT assay. Data are shown as mean ± SD from three 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 four models. Based on in vitro potencies, sunitinib was included as a second comparator in two models (Δ557-558/V654A and Δ557-558/T670I) in which imatinib was expected to be non-efficacious, 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 to the relevant vehicle group using one way ANOVA, with statistical significance (p< 0.05) indicated by an asterisk.
Figure 2
Figure 2. Secondary Resistance Mutants Identified in the Presence of KIT Inhibitors
Resistant cells recovered from N-ethyl-N-nitrosourea treated Ba/F3 KIT Ex11 (Δ557-558) cells, 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 three separate studies.
Figure 3
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 referenced in the text in green, the A-loop in cyan and the 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
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 due to >20% body weight loss.
Figure 5
Figure 5. Single Agent Ponatinib is Active in Heavily Pretreated, TKI Resistant GIST Patients
Representative CT scans of three KIT exon 11-mutant GIST patients before and after treatment with 30 mg ponatinib for four 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, while yellow arrows indicate tumor response.

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