In vitro and In vivo study of the Efficacy of Cabozantinib on various FLT3 mutations
Date Issued
2016
Date
2016
Author(s)
Liu, Zheng-Hau
Abstract
FLT3 is expressed by hematopoietic stem cells and progenitor cells and its function is to regulate proliferation and differentiation. In clinical studies, about one third of acute myeloid leukemia (AML) patients have mutation on FLT3, and the majority of mutation on FLT3 is FLT3-internal tandem duplication (ITD). Patients with FLT3/ITD have poor prognosis, high risk of relapse and decreased survival. FLT3-ITD is a driver mutation, so FLT3 is considered to be a target for therapies. In our previous study, we found that cabozantinib , a small molecule kinase inhibiter for MET, VEGFR2, RET ,KIT, and FLT3, is an effective inhibitor for FLT3/ITD AML cell growth both in vivo and in vitro. To confirm the selective cytotoxicity to FLT3/ITD AML cell line of cabozantinib in vivo, we used MV4-11, Molm13, U937, OCI-AML3 xenograft model by subcutaneous injection into nude mice. We found that only AML cell line harboring FLT3/ITD had decreased tumor growth and improved survival after cabozantinib treatment. However, emergence of drug resistance after small molecule kinase inhibitor treatment is a big challenge for treatment. That is the reason why studying on drug resistance of cabozantinib is needed. In present study, we focus on the AML cells that express various FLT3-tyrosine kinase domain (TKD) mutations, and found that they had various drug responses and might have drug resistance to tyrosine kinase inhibitor. We chose four common TKD mutations including F691L, N676D, D835Y and Y842H in our study. Tyrosine kinase domain mutations on p-EGFP N3-FLT3/ITD plasmid were introduced by site-directed mutagenesis and then confirmed that all FLT3/ITD and tyrosine kinase domain mutation base were correct by DNA sequencing. We transfected FLT3/ITD-TKD mutation to 32D cells by using electroporation. FLT3/ITD-TKD expression was checked with RT-PCR and cDNA sequencing. By using MTS assay, we found that mutations on tyrosine kinase domain 1 (F691L, N676D) had better response to cabozantinib than those mutations on tyrosine kinase domain 2 (D835Y, Y842H ). We also used Western blot to confirm the efficacy of cabozantinib on various TKD mutations. We found that various TKD mutation had different response to cabozantinib, and those molecule associated with downstream signaling pathway of FLT3 were inhibited in higher concentration of cabozantinib treatment. Finally, we tried to build 32D animal model for AML. First, we found that the weight of C3H/HeNCrNarl mice were not affected after 10 mg/kg cabozantinib treatment for seven days. Subsequently, we used tail vein injection to test when the 32D cells could induce leukemia phenotype in C3H/HeN mice, and we found seven of twelve C3H/HeN mice had leukemia phenotype. Their course of disease was about 70 to 100 days. In conclusion, cabozantinib is effective to inhibit tumor growth of FLT3-ITD AML cell line in vitro and in vivo. We also built a FLT3-ITD-TKD 32D animal model for AML by tail vein injection. Their incidence is about 60%.
Subjects
Acute myeloid leukemia
TKD mutation
Type
thesis
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