In vitro and In vivo study of the Efficacy of Cabozantinib on various FLT3 mutations

FLT3是一個表現在造血幹細胞與造血前驅細胞的一個接收器，其作用是調節細胞增生與分化。而在臨床的研究發現，大約三分之一的急性骨髓性白血病病患都帶有FLT3的突變，其中最主要的突變就是FLT3-ITD(內部串聯重複)。帶有FLT3-ITD突變的急性骨髓性白血病病患往往預後比較差、高復發風險且有較低的存活率；由於FLT3-ITD在白血病的發展上扮演驅動突變(driver mutation)的角色，因此FLT3儼然成為一個發展治療藥物的重要標的。在實驗室先前的研究發現，一種抑制MET、VEGFR2、RET、KIT及FLT3的小分子激酶抑制藥物；cabozantinib，能有效的抑制帶有FLT3-ITD的急性骨髓性白血病細胞株的細胞生長。為了確認此藥物在活體實驗中，對於帶有FLT3-ITD的急性骨髓性白血病細胞株有選擇性的毒殺。我們採用了MV4-11, Molm13, U937, OCI-AML3四種皮下注射的異種移植動物模型，我們發現只有帶有FLT3-ITD的細胞株MV4-11, Molm13會受到藥物影響而減少其腫瘤生長，並且改善其異種移植小鼠存活率。 然而，對於小分子標靶藥物治療最大的挑戰還是抗藥性的問題；因此，研究cabozantinib可能遇到的抗藥性問題是必須的。在先前的文獻指出，急性骨髓性白血病若發生了酪氨酸激酶結構域(TKD)的結構突變，不同的突變位點會對不同藥物有不一樣的感受性，甚至會有抗藥性的問題。為了探究這個問題我們選用了四種常見的TKD突變包括F691L、N676D、D835Y、Y842H，我們利用定點突變將帶有FLT3-ITD的pEGFP N3載體放入TKD的點突變；接著利用DNA定序來確認所有序列正確且有成功完成點突變。利用電穿孔的方式將FLT3-ITD-TKD 突變送入32D細胞株後，利用RNA的萃取，並反轉錄成cDNA，然後送定序確認其序列。利用MTS assay來測試藥物對細胞的IC50，結果我們發現在TKD1點突變(F691L、N676D)的細胞，比起在TKD2點突變(D835Y、Y842H)的細胞，對於cabozantinib仍具有感受性。我們也利用了西方墨點法來確認cabozantinib對於不同的TKD突變的訊息傳遞抑制效果；我們發現不同的TKD突變會有不同的藥物感受性。 最後，我們建立FLT3-ITD 32D誘導的急性白血病動物模型。在予以10mg/kg cabozantinib七天後，所使用的C3H/HeNCrNarl 小鼠不會有體重的降低或其他不適症狀。運用鼠尾靜脈注射方式，將帶有各種FLT3-ITD的32D細胞注入鼠尾靜脈。我們發現十二隻中有七隻的C3H/HeN小鼠有成功誘導出急性骨髓性白血病，發病的時間約七十到一百天。 本論文顯示除了細胞實驗中cabozantinib能有效抑制具有FLT3-ITD細胞的生長之外，也能在動物實驗中得到類似的結果。另外，也建立一個鼠尾靜脈注射帶有FLT3-ITD的32D細胞株誘導AML的實驗動物模型，發病率約60%。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%