Cabozantinib對FLT3-ITD之急性骨髓性白血病細胞具有選擇性毒殺效果

Abstract

FLT3內部串聯重複突變(FLT3-ITD)為AML最常見的突變之一，發生率約30%，臨床上帶有FLT3-ITD的AML病患預後不佳，因此FLT3-ITD被認為是很有潛力的治療標的。Cabozantinib (XL-184)為一口服的多激酶小分子抑制劑，可以抑制與癌症致病相關之MET、VEGFR2、RET、KIT、TIE-2以及FLT3的活性。 首先在細胞抑殺試驗(MTS assay)中，我們發現帶有FLT3-ITD的細胞株 MV4-11及MOLM-13對cabozantinib的感受性極高 (IC50 < 5 nM)。低濃度的cabozantinib對MV4-11細胞生長和FLT3及其下游訊息傳遞路徑之活化有抑制效果。利用流式細胞分析儀分析，發現cabozantinib會以dose-dependent的方式造成MV4-11及MOLM-13細胞停留在細胞週期中的G0/G1期，並引發細胞凋亡。於西方墨點法分析中也可以偵測到活化態的Caspase 3及PARP-1蛋白，亦證明cabozantinib能使MV4-11細胞走向細胞凋亡。進一步分析與細胞凋亡相關蛋白，顯示cabozantinib處理會down-regulate抗凋亡蛋白Survivin、Mcl-1及up-regulate促凋亡蛋白Bak之表現，尤其以Survivin表現量的變化最為明顯。 Cabozantinib除了對具有FLT3-ITD之血癌細胞株具有毒殺效果，我們發現cabozantinib亦能明顯抑制FLT3-ITD之AML病患骨髓單核細胞的存活。 目前斑馬魚已逐漸成為毒物測試及藥物篩選之實驗動物模式的另一種選擇。我們利用異種移植斑馬魚測試cabozantinib於in vivo的藥效，結果顯示cabozantinib低毒性且具有清除MV4-11細胞的能力。 總體而言，我們發現cabozantinib對FLT3-ITD AML細胞具有選擇性毒殺效果，藉由充分抑制FLT3異常活化而抑制AML細胞生長並促進細胞凋亡。

FLT3 internal tandem duplication (ITD) mutations are found in approximately 30% of acute myeloid leukemia (AML) patients and are associated with poor prognosis. FLT3-ITD is therefore a potential target for therapy. Cabozantinib (XL-184) is an oral multikinase inhibitor that targets MET, VEGFR2, RET, KIT, TIE-2, and FLT3, all of which have been implicated in tumor pathogenesis. We found both of the FLT3-ITD cell lines, MV4-11 and MOLM-13, were extremely sensitive to cabozantinib with IC50 of less than 5 nM by MTS assay. In MV4-11 cells, low concentration of cabozantinib treatment resulted in decreased cell proliferation and potent inhibition of FLT3 phosphorylayion and downstream signaling. G0/G1 cell cycle arrest and induction of apoptosis in a dose-dependent manner were observed upon treatment with cabozantinib in MV4-11 and MOLM-13 cells by flow cytometry analysis. Cleavage of Caspase 3 and PARP-1 were also observed in cabozantinib treated MV4-11 cells by western blot analysis. Further investigations on apoptosis-related proteins revealed that cabozantinib induced apoptosis through down-regulation of anti-apoptotic proteins Survivin and Mcl-1 and up-regulation of pro-apoptotic protein Bak. We also determined the sensitivity of primary bone marrow mononuclear cells to cabozantinib ex vivo by MTS assay. A significant reduction of cell viability was observed in FLT3-ITD AML cells. Zebrafish has emerged as an alternative model organism for toxicity testing and drug screening in vivo. Exposure of xenograft zebrafish to cabozantinib demonstrated low toxicity and a considerable anti-leukemic activity. Taken together, these results show that cabozantinib potently inhibited the phosphorylation of FLT3 and selectively induced cell cycle arrest and apoptosis in FLT3-ITD AML.