Cabozantinib抗藥性血癌細胞株Molm13-XR的建立、特性分析以及治療策略

Abstract

FLT3內部串聯重複(FLT3-ITD)是急性骨髓性白血病常見的突變類型，約有30~40%的AML病人具有此突變，這類的病患在臨床上的預後不佳;因此，FLT3被認為是有潛力的治療標的，目前也已經發展出許多能夠作用於FLT3的標靶藥物，其中包括cabozantinib(XL184)。 實驗室先前在in vitro的研究中發現cabozantinib於低劑量即能夠抑制FLT3-ITD突變的AML細胞株Molm13，在in vivo實驗亦看到cabozantinib能夠顯著地抑制Molm13腫瘤的生長。由於使用FLT3標靶藥物常會有復發的情形，因此有必要著手進行Molm13對於cabozantinib的抗藥性探討。 本篇研究利用持續給予藥物的方式，為期5個月後建立出對cabozantinib具抗藥性的細胞株Molm13-XR，其對於cabozantinib的IC50為473.36 ±154.73 nM，母細胞株Molm13-P為1.06±0.93 nM。以RNA-seq篩選後發現Molm13-XR具有FLT3 D835Y突變，此結果也進一步以pyrosequencing確認。 另外以phospho-kinase array篩選並用西方墨點法確認FLT3以及其下游訊息傳遞路徑表現情形，觀察到p-FLT3、p-AKT、p-STAT5以及p-ERK皆增加。此外，phospho-kinase array的篩選中發現Molm13-XR的p-GSK3β-Ser9在眾多激酶當中有最大程度的增加且以西方墨點法確認，並觀察到下游的抗凋亡蛋白Mcl-1增加。 其他藥物的細胞抑制實驗結果顯示Molm13-XR對於第一型的FLT3抑制劑midostaurin以及crenolanib仍具有感受性，能夠在低濃度下有抑制效果;然而對於第二型的FLT3抑制劑sorafenib、quizartinib則依然具有抗藥性。除了FLT3抑制劑之外，還另外使用了MEK抑制劑CI-1040、AKT抑制劑MK-2206，但皆無法在臨床上可達到的藥物濃度下達到抑制效果。 Pyrvinium pamote(PP)是一種FDA已核准使用的蟯蟲治療用藥。我們發現其於低濃度下即可以抑制不論是Molm13-P或是Molm13-XR的生長，進一步分析其能夠引發細胞凋亡、減少細胞的ATP。另外以西方墨點法偵測到AKT、STAT5的活性受到抑制，以及抗凋亡蛋白Mcl-1的減少、切割態的caspase 3增加。 總體而言，從我們的實驗結果顯示FLT3 D835Y以及抗凋亡蛋白Mcl-1增加，可能是造成Molm13-XR對cabozantinib產生抗藥性的原因。

Internal tandem duplication mutation in FLT3 (FLT3-ITD) represents one of the most common gene alterations in AML and confers a poor prognosis. FLT3 has emerged as a promising molecular target in therapy of AML. There are several FLT3 inhibitors have been used in clinical trials including cabozantinib (XL184). Our previous work demonstrated that cabozantinib potently inhibited the viability of a FLT3-ITD+ Molm13 cell line also known as Molm13-P in vitro. Also, cabozantinib significantly delayed the outgrowth of Molm13-P tumor compaired with vehicle treated mices in vivo. However, drug resistance in patients after target therapy has become a clinical problem. Therefore, it is necessary to understand the underlying mechanisms confering drug resistance. We generated cabozantinib resistant Molm13 cells also known as Molm13-XR by treating Molm13-P with progressively increasing concentrations of cabozantinib from 1nM to 1μM for approximately 5 months. Cabozantinib potently decreased the viability of Molm13-P with a 50% inhibitory concentration (IC50) of 1.06±0.93 nM, whereas the resistant strain Molm13-XR was 473.36 ±154.73 nM. There was a new FLT3 D835Y mutation in Molm13-XR screened by RNA-seq and confirmed by pyrosequencing. In addition, phosphorylated level of FLT3 and its downstream molecules were screened by phospho-kinase array and confirmed by western blot. We demonstrated that there were increased phosphorelated level of FLT3、AKT、STAT5 and ERK. Morever, p-GSK3β-Ser9 had the most increased level among all kinases in Molm13-XR compared with Molm13-P screened by phospho-kinase array and confirmed by western blot. Furthermore, there was higher level of Mcl-1, an anti-apoptotic protein, in Molm13-XR compared with Molm13-P. By performing cytotoxicity assay in Molm13-XR, our results suggested Molm13-XR exhibited good susceptibility to midosaturin and crenolanib while remaning resistant to sorafenib and quizartinib. Other than FLT3 inhibitors, we also tested MEK inhibitor, CI-1040、AKT inhibitor, MK-2206. However, these inhibitors couldn’t inhibit Molm13-XR at clinically releveant doses. We found that pyrvinium pamote(PP), an anthelmintic drug approved by FDA, had a potent cytotoxicity toward Molm13-P and Molm13-XR at a low nanomolar concentration of 14.81±10.77 and 13.45±6.59 nM. PP would inhibit AKT and STAT5 activities and decrease ATP level in Molm13-XR. Also, PP would downregulate anti-apoptotic protein Mcl-1 and upregulate cleaved caspase 3 in Molm13-XR. PP would induce apoptosis and decrease ATP level in Molm13-XR. The AKT and STAT5 signaling were downregulated and anti-apoptotic protein Mcl-1 is decreased by pyrvinium pamote treatment. Overall, we established a cabozantinib resistant AML cell line Molm13-XR. Our results show that FLT3 D835Y mutation and upregulation of Mcl-1might contribute to cabozantinib resistance.