A Potent Small Molecule Compound Might Overcome High Level Drug Resistant Mutations of Bcr-Abl1

Objective: The third-generation tyrosine kinase inhibitor (TKI) ponatinib can overcome many kinds of single mutations including T315I of Bcr-Abl1. Unfortunately, recent clinical studies have identified some new point mutations resistant to ponatinib and the other marketed TKIs. These mutations include compound mutations or T315M single mutation, which we called high level drug resistant mutations in this study. Therefore, it is an urgent task to find a potent small molecule inhibitors, which can overcome these high level drug resistant mutations.

Methods: To screen for compounds that can overcome the high level drug resistant mutations of Bcr-Abl1, we firstly established genetic engineering cell models using the Ba/F3 cell line, including: (1) Ba/F3-Bcr-Abl1^T315M; (2) Ba/F3-Bcr-Abl1^T315I/E255V; (3) Ba/F3-Bcr-Abl1^E255V/Y253H. The established cell lines were also carefully verified .Then we used these established cell models and others we have already obtained, including Ba/F3-Bcr-Abl1^WT, Ba/F3-Bcr-Abl1^T315I, and Ba/F3-Bcr- Abl1^E255V, to screen an in-house chemical library. The most potent compound SKLB-518 was finally chosen to perform further studies both in vitro (cell and protein level) and in vivo (allograft subcutaneous cancer mice model).

Results:In vitro assays, three marketed TKIs, namely imatinib, dasatinib, and ponatinib, were chosen as the references. The IC50 value of SKLB-518 to inhibit Ba/F3-Bcr-Abl1^E255V/Y254H cells was 0.033 μM, which was more active by >100 times and 6.14 times than that of imatinib (IC50: >10 μM) and ponatinib (IC50:0.201μM), respectively, and comparable with dasatinib (IC50:0.028 μM). The IC50 of SKLB-518 to inhibit Ba/F3- Bcr-Abl1^T315I/E255V cells was 0.094 μM, which was more potent by >100 times, 30~100 times, and 8.69 times than that of imatinib (IC50: >10 μM), dasatinib (IC50: 3~10 μM), and ponatinib (IC50: 0.817 μM), respectively. In vivo, thedata indicated that SKLB-518 could significantly inhibit tumor proliferation at the doses of 30 mg/kg/d and 45 mg/kg/d (%T/C was 62.1 and 72.5, respectively; both groups p<0.001), while ponatinib couldn't inhibit tumor growth at the 30 mg/kg/day dose (%T/C was 87.8, p=0.157). Immunohistochemical staining assays indicated that SKLB-518 at the doses of 45 mg/kg/d could potently inhibit Bcr-Abl1 downstream targets (Crkl and Stat5). Meanwhile, we didn't find severe toxicity of SKLB-518 in the effective doses both in cells and in mice.

Conclusion:Independently developed small molecule SKLB- 518 might overcome the high level drug resistant mutations of Bcr-Abl1.