摘要:Cabozantinib是一種具有抗癌功能的口服多磷酸酶抑制劑。美國食品藥物管理局已經在2012年通 過它適用在轉移曱狀腺髓質癌;另外在2015年也接受適用在惡性腎細胞癌的申請。我們已經在細胞 實驗上發現cabozantinib能夠針對具有f/J3-ITD的MV4-11及MOLM13血癌細胞之生長有很好的抑制 作用,IC50分別為2.4 nM及2.0 nM,遠低於血漿中的濃度。進一步老鼠實驗中也證實給予低劑量 10mg/kg就能有效抑制MV4-11及MOLM-13腫瘤的生長並有較高的存活率;顯示Cabozantinib值得 進一步進行急性骨髓性白血病的臨床實驗。不過,還有一些抗藥性相關的議題需要加以釐清,以期更 能精準的選取適當的病患進入臨床試驗。目前關於口服FLT3抑制劑在具有f/J3-ITD的患者上最大的缺陷是服用一陣子之後會有抗藥性的產 生。在抗藥細胞上常出現原先細胞沒有的基因變異在tyrosine kinase domain上;而不同的多磷酸酶抑 制劑所產生的基因變異位置不盡相同。過去的研究報告指出Cabozantinib對於與曱狀腺及腎細胞相關 的RET及MET的基因變異(分別為M918T, C634W與Y1248H, D1246N, K1262R)仍具有效的抑制能力。 因此,我們預期cabozantinib對一些FLT3抑制劑所產生的FLT3的基因變異(例如fLT3-ITD+F691L, FLT3-ITD+N676D, FLT3-ITD+Y842H 及其他)是會有效的。(目標一)因為抗藥性的產生是無可避免的;因此,我們也同時著手cabozantinib抗藥性的研究。我們長時 間將MV4-11培養在具有自低劑量緩升到高劑量的cabozantinib培養液中,得到IC50上升到1298.4nM 的MV4-11-XR細胞。進一步FLT3基因序列分析的結果顯示除了之前在其他藥物有被報告過的 FLT3-ITD+D835Y外,還有未曾被報告過之fLT3-ITD-E20del的基因變異出現。因此,進一步的探究 FLT3-ITD-E20del的生物意義、造成抗藥性的機制以及尋找適當其他FLT3抑制劑的配合都是值得去進行 的。(目標二)除了 FLT3基因變異之外,我們也分析到與MV4-11相比,MV4-11-XR細胞之FJ3-ITD相關的訊息傳遞路徑上的一些分子磷酸化的變化:在FLT3磷酸化仍被抑制的情況下,其下游的STAT5、AKT與ERK 磷酸化仍然高居不下。因此,從非基因變異的抗藥機制上面去探究,結合不同屬性的抑制劑、結合 cabozantinib與下游抑制劑、同時給藥或依序給藥等方式克服抗藥,都是值得去進行的。(目標三) 近年來,我們實驗室也致力於建立轉殖斑馬魚在骨髓性白血病上的研究平台,成果之一就是 FLT3-ITD轉殖斑馬魚在9個月大時有骨髓細細胞增生及分化受阻的表象;顯示轉殖斑馬魚是值得運用 在骨髓性白血病的研究利器。因此,在此三年計劃中,針對前述的研究方向,我們將從in vitro及in vivo 雙方面釐清cabozantinib在急性骨髓性白血病的使用效力以及運用策略。In vitro方面主要是運用 cabozantinib抗藥細胞株MV4-11-XR以及外送各種FLT3-ITD+TKD基因到32D細胞來進行各細項的分析; 另一方面in vivo則包括建立轉殖基因斑馬魚平台作深入的探討。這些研究成果將完整釐清cabozantinib 針對各種FLT3基因突變之血癌細胞的作用機制以及抗藥機制;並提供未來臨床上針對具有FLT3基因 變異的急性骨髓性白血病患者在各種標靶治療用藥上面的運用策略。
Abstract: Cabozantinib is an oral multikinase inhibitor that exhibits anti-tumour activity in several cancers. The US Food and Drug Administration (FDA) approved cabozantinib for the treatment of progressive metastatic medullar thyroid cancer in 2012, and accepted the submission for treating advanced renal cell carcinoma in 2015. We found that cabozantinib was significantly cytotoxic to MV4-11 and Molm-13 cells that harboured FLT3-ITD, resulting in IC50 values of 2.4 nM and 2.0 nM, respectively. In a mouse xenograft experiment, we revealed that cabozantinib significantly inhibited MV4-11 and Molm-13 tumour growth at dosage of 10mg/kg and demonstrated a longer survival rate, suggesting clinical trials evaluating the efficacy of cabozantinib in acute myeloid leukaemia (AML) with FLT3-ITD are warranted. However, some issues associated with drug resistance needs to be clarified before recruiting proper patients to enter these kinds of clinical trials.It is also known that the most frequent mode of acquired resistance is the acquisition of point mutations in the tyrosine kinase domain (TKD) and the resistance profiles that emerge are non-overlapping. Cabozantinib has been reported to be a potent inhibitor of RET and MET, and demonstrates efficacy on cells with various RET mutations (M918T, C634W) and MET mutations (Y1248H, D1246N, and K1262R). It is known that resistance to FLT3 inhibitors has emerged as a common theme. Thus, the efficacy of cabozantinib against common drug resistance FLT3 mutation, including, but not limited to, FLT3-ITD-F691L, FLT3-ITD-N676D, and FLT3-ITD-Y842H could also be expectable. (Aim 1)In order to elucidate the drug resistance of cabozantinib, we established a cabozantinib-resistant MV4-11 cell line (MV4-11-XR) and examined some characteristics of them. Direct sequencing analysis demonstrated FLT3-ITD-D835Y and FLT3-ITD-E20del emerged in MV4-11-XR. Of them, FLT3-ITD-E20del is a novel FLT3-TKD mutation. Consequently, further investigation is needed to determine the biologic significance and potential mechanistic underpinning of these FLT3 mutations in the development of resistance to cabozantinib. (Aim 2)Compared to parental MV4-11 cells, MV4-11-XR cells had more prominent phosphorylated STAT5, Akt and ERK. Thus, rational combinations of other compounds or switching FLT3 inhibitors could be expectable for overcoming this resistance. (Aim3)Therefore, in this three-year proposal, we will elucidate the efficacy of cabozantinib in AML stated above from both in vitro and in vivo aspects. MV4-11-XR and 32D cells harbouring various FLT3-ITD+TKD mutations will be used for in vitro study. Transgenic zebrafish with various FLT3-ITD+TKD mutations will also be used for in vivo study based on our previous experience on FLT3-ITD transgenic zebrafish with phenotype of myeloid expansion and poor differentiation at age of 9 months. These results will clarify the molecular mechanism of cabozantinib targeting leukemic cells with various FLT3-ITD+TKD mutations and the cabozantinib-resistance. In addition, therapeutic strategy with various combinations will be planned to overcome drug resistance.