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. 2019 Mar 16;9(7):1952-1964.
doi: 10.7150/thno.30890. eCollection 2019.

Repurposing Ponatinib as a Potent Agent against KIT Mutant Melanomas

Yong Han  1   2   3 , Ziyue Gu  1   2   3 , Jing Wu  1   2   3 , Xiaojuan Huang  1   2   3 , Rong Zhou  1   2   3 , Chaoji Shi  2 , Wenjie Tao  1   2   3 , Lizhen Wang  4 , Yanan Wang  1 , Guoyu Zhou  1 , Jiang Li  4 , Zhiyuan Zhang  1   2   3 , Shuyang Sun  1   2   3
Affiliations

Repurposing Ponatinib as a Potent Agent against KIT Mutant Melanomas

Yong Han et al. Theranostics. .

Abstract

Rationale: Mutations in KIT, a major cancer driver gene, are now considered as important drug targets for the treatment of melanomas arising from mucosal and acral tissues and from chronically sun-damaged sites. At present, imatinib is the only targeted drug for KIT-mutation-bearing melanomas that is recommended by the National Comprehensive Cancer Network (NCCN) Clinical Practice guidelines. Patients with KIT mutations, however, are either insensitive or rapidly progress to imatinib insensitivity, which restricts its clinical use. Thus, effective inhibitors of KIT-mutation-bearing melanomas are urgently needed. Methods: A cohort of patient-derived tumor xenograft (PDX) models and corresponding PDX-derived cells (PDCs) from patients with melanomas harboring KIT mutations (KITV560D, KITK642E and KITD816V) were established, characterized, and then used to test the in vitro and, subsequently, in vivo inhibitory effects of a panel of known KIT inhibitors. Results: Ponatinib was more potent than imatinib against cells bearing KIT mutations. In vivo drug efficacy evaluation experiments showed that ponatinib treatment caused much stronger inhibition of KIT-mutation-bearing melanomas than did imatinib. Mechanistically, molecular dynamics (MD) simulations revealed a plausible atomic-level explanation for the observation that ponatinib has a higher affinity for the KITD816V mutant protein than does imatinib. Conclusions: Our study of KIT-mutation-and KITWT-bearing melanomas demonstrates that ponatinib is a far more potent inhibitor than is imatinib for KIT-mutation-bearing melanomas and thus underscores that ponatinib should be given priority consideration for the design of precision treatments for melanoma patients triaged to have KIT mutations. Moreover, our work provides a rationale for undertaking clinical trials to examine the repurposing of ponatinib, which is already approved for use in leukemia, for use in treating a large subset of melanoma patients.

Keywords: KIT; melanomas; patient-derived xenograft models; ponatinib.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Establishment and characterization of KITV560D PDX models. (A) Schematic of PDX and PDC establishment. (B) Representative hematoxylin and eosin (H&E) staining and immunofluorescence staining of patient tumors and corresponding PDX model tumors. HMB-45 and Melan-A (both green), Ki-67 (red) and DAPI (blue). The scale bar is 100 μm. (C) Representative immunofluorescence staining of patient-derived tumor cells from KITV560D mutant melanoma PDX models are shown in the upper panel, and the KIT mutation status of the corresponding PDX-derived cells is shown in the bottom panel. HMB-45, Melan-A and S-100 (both green), and DAPI (blue). The scale bar is 100 μm.
Figure 2
Figure 2
In vivo evaluation of imatinib efficacy in KITV560D and KITK642E mutant PDX models. (A-B) Inhibition efficacy of imatinib in PDX models with KITV560D mutation (n= 5 mice per group). Imatinib, 50 mg/kg, tumor growth inhibition, TGI= 25.04%, P>0.05; imatinib, 100 mg/kg, TGI= 35.81%, P >0.05, Student's t-test. (C-D) Inhibition efficacy of imatinib in PDX models with KITK642E mutation (n=6 mice per group). Imatinib, 100 mg/kg, TGI = 35.81%, Student's t-test, P > 0.05.
Figure 3
Figure 3
In vitro drug efficacy evaluation using KITWT, KITV560D, KITK642E and KITD816V PDX-derived cells. (A-D) Dose-response curve of imatinib, dasatinib, axitinib, nilotinib, ponatinib, sorafenib, and sunitinib in 72-h proliferation assays with KITWT, KITV560D, KITK642E and KITD816V PDX-derived cells. (E-F) Immunoblot analysis of KIT signaling in KITWT, KITV560D, KITK642E and KITD816V PDX-derived cells treated with imatinib, dasatinib, or ponatinib for 2 hours.
Figure 4
Figure 4
In vivo drug efficacy evaluation in KITWT, KITV560D, KITK642E and KITD816V mutant PDX models. We administered imatinib (100 mg/kg), dasatinib (30 mg/kg) and ponatinib (30 mg/kg) to PDX mice daily for 28 days. Tumor volume and tumor weight were measured, and the results are summarized as the mean ± SEM (Student's t-test). Representative tumor images of these experiments (taken on day 28). (A-C) PDX-KITWT, treated with imatinib (TGI = 17.96%), dasatinib (TGI = 33.85%), and ponatinib (TGI= 33.26%), n = 6. (D-F) PDX with KITV560D mutation, treated with imatinib (TGI = 25.25%), dasatinib (TGI = 68.65%), and ponatinib (TGI = 78.33%), n = 6. (G-I) PDX with KITK642E mutation, treated with imatinib (TGI = 27.59%), dasatinib (TGI = 81.38%) and ponatinib (TGI = 83.66%), n = 5. (J-L) PDX with KITD816V mutation, treated with imatinib (TGI = 42.67%), dasatinib (TGI = 67.73%) and ponatinib (TGI = 99.95%), n = 5 *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant; n indicates the number of tumors per arm.
Figure 5
Figure 5
Inhibitory efficacy of ponatinib in PDX with KITD816V mutation. (A) Immunoblot analysis of KIT signaling in tumors after 28 days of treatment with imatinib, dasatinib and ponatinib. (B) Scoring of Ki-67 staining is summarized as the mean ± SEM. Student's t-test, **, P < 0.01; ***, P< 0.001. (C) Scoring of TUNEL staining is summarized as the mean ± SEM. Student's t-test, ***, P < 0.001. (D) Representative Ki-67 staining in tumors after 28 days treatment with imatinib, dasatinib and ponatinib. Scale bar, 50μm. (E) Representative TUNEL staining in tumors after 28 days of treatment with imatinib, dasatinib and ponatinib. Scale bar, 50μm.
Figure 6
Figure 6
RNA-sequencing in tumors of PDX-KITK642E after 28 days of treatment with imatinib (n = 3) and ponatinib (n = 3). (A) Heatmap of differential expressed genes between the imatinib-treated group and ponatinib-treated group. (B) KEGG analysis showed that the ECM-receptor interaction pathway was the most significantly downregulated pathway in ponatinib-treated tumors compared with imatinib-treated tumors.
Figure 7
Figure 7
Molecular dynamic simulations of KITWT/ KITD816V-ponatinib interactions. (A) RMSD of KITWT/ KITD816V-imatinib complexes along 100 ns of MD simulations. (B) RMSD of KITWT/ KITD816V-ponatinib complexes along 100 ns of MD simulations.
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